Human antibodies specific to an invasion ligand may play an important role in immunity. Comparisons between antibody levels to PfRh4.9 in samples from children and the concentration of affinity-purified antibodies for invasion inhibition suggested that protected children have sufficiently high antibody levels to have inhibitory activity against P. falciparum. These data complement our findings of an association between antibodies to PfRh4 and protection from malaria, providing evidence that PfRh4 is a Benzethonium Chloride target of protective antibodies. In contrast, human PfRh4.2 antibodies did not inhibit invasion of erythrocytes. Hence, the functional relevance of PfRh4.2 antibodies is unclear, and they may have a 4-(Benzyloxy)phenol different mode of action, such as interactions with Fc-receptors on immune cells or with complement. Alternatively, antibodies to PfRh4.2 may be a marker of immunity, or of responses to PfRh4 more broadly, rather than directly contributing to immunity. Presently, the acquisition of human growthinhibitory antibodies to merozoite antigens has only been demonstrated for AMA1 and MSP1-19, and with some evidence supporting EBA175 as an additional target. Studies have not previously related the antibody levels required for functional activity to levels present in protected individuals as we have done. Prior studies suggest that MP1-19 is not a major target of inhibitory antibodies in this population. Most isolates infecting children demonstrated PfRh4 protein expression, consistent with our data suggesting PfRh4 may be an important target of acquired immunity. There are no prior reports of PfRh4 protein expression in clinical isolates, but two studies of African children found that pfrh4 gene expression was detected in a minority of isolates. Differences between these studies and ours may represent population-specific differences, or differences in the sensitivity of the methods used. Consistent with our studies on clinical isolates, most culture adapted isolates show expression of PfRh4 protein. Further studies are needed to assess a larger number of isolates in different populations and relationship between PfRh4 expression and acquired antibodies. Sequence analysis identified little polymorphism in the PfRh4 erythrocyte-binding region and no polymorphisms in the PfRh4.2 region. In contrast, PfRh2 has a highly polymorphic stretch in the N-terminal region that encodes the erythrocyte-binding domain. It is possible the variable expression of PfRh4 is used a means of immune evasion, rather than immune evasion via the evolution of polymorphisms. The limited polymorphism in PfRh4 may make it attractive as a vaccine candidate, especially when partnered with other antigens, such as EBA175, to block the use of alternate invasion pathways. PfRh5 also has limited polymorphisms, and a recent report suggested that there was minimal acquisition of IgG to recombinant PfRh5 in a population of malaria-exposed Kenyans. One study reported that expression of the pfrh5 gene was variable among P. falciparum isolates in The Gambia, as seen for other pfrh genes, whereas other data suggests PfRh5 protein is expressed by all laboratory-adapted isolates. Our preliminary studies suggest that the prevalence of antibodies to recombinant PfRh5 is high in our PNG study population. Further studies to understand the expression of PfRh ligands and the acquisition of antibodies to them will be valuable for understanding the importance of PfRh proteins as targets of immunity and their potential for vaccine development. Understanding the targets of acquired human immunity is valuable for several reasons. One of the important criteria for objectively prioritizing antigens for vaccine development against malaria, as with other infectious pathogens, is the demonstration that immune responses to that antigen are associated with protection from malaria.