In the present paper we provide evidence that CT and LT ADP-ribosylated defensins, which thus represent novel substrates for these bacterial ARTs. On the other hand, NarE and ExoS did not modify either a- or bdefensins. Interestingly, unmodified HNP-1 exerted inhibition on NarE transferase activity suggesting a regulatory role. While the ADP-ribosyltransferase activity was inhibited by HNP-1, the NAD-glycohydrolase activity remained CUDC-907 unaltered. Furthermore, HNP-1 strongly enhanced the auto-ADP-ribosylation of NarE, a recently discovered catalytic activity of this toxin. Overall, our data highlight the interplay between ADP-ribosylating toxins and human defensins. ADP-ribosylating toxins are usually secreted by bacterial pathogens in the host environment. Some of them, which possess arginine-specificity, could recognize arginine-rich peptides such as a- and b- defensins as substrates. Both a- and b- defensins are released by neutrophils and epithelial cells respectively in high amounts at inflammatory sites. In this report we present evidence that synthetic HNP-1 and HBD1 are ADP-ribosylated in vitro by CTA and LTA. In contrast they are not recognized as substrates by ExoS and only poorly by NarE, suggesting specificity for both bacterial toxins and substrates. The artificial kemptide, which contains a di-arginine motif, was modified by CT on the first arginine of the motif while a mammalian ART recognized the second arginine within the R-R motif. In contrast, our data indicate R14 as the preferred modification site, since the HNP-1-R14K was not ADP-ribosylated by the ADP-ribosylating toxins used in this study. Our findings are in contrast with studies performed by other groups, which failed to show toxin-catalyzed incorporation of the ADPribose unit on defensins. Others evaluated the presence of the ADP-ribosylated-HNP-1 by monitoring the absorbance of the modified peptide in reversed-phase chromatography, but not being successful in identifying it. Therefore we chose a chemiluminescence assay to detect ADP-ribosylation because of the higher sensitivity, allowing the detection of small amounts of modified HNP-1. In agreement with previous findings we did not observe incorporation of ADP-ribose with PT and ExoS. Labelling of proteins can also result from the covalent noncatalyzed reaction of NAD or free ADP-ribose with the eamino groups of lysines. ADP-ribosylation of HNP-1, in which lysine residues are absent, was not blocked by the addition of free ADP-ribose, while a reduction of incorporation was noticed when unlabelled NAD was added to the reaction mixture. Comparable results were obtained with HBD1 that contains four lysine residues. These data, further supported by mass spectrometry analysis, strongly indicate that an enzymatic ADP-ribosylation, and not a secondary reaction with NAD or ADP-ribose, was responsible for the modification. Defensins belonging to a- and b- group contain several conserved arginines, which are recognized by CT and LT. However they are devoid of diphtamide and asparagine residues, which are the target amino-acid of DT, ETA and clostridial toxins. Furthermore, cysteines, present in a- and b- defensins and recognized by PT as ADP-ribose acceptors, are engaged in disulphide bridges. It is well known that defensins have a variety of activities, but the antimicrobial function is by far the most important. Thus ADPribosylation of selected arginines might well correlate with recent discoveries, which show that antibacterial activity strictly depends on cationicity and that only selective arginines support this activity. Interestingly, toxic activities are not decreased in the case of CT, which ADP-ribosylates HNP-1 at an arginine residue.