Neurodevelopmental disorder in which the lateral rectus muscle, responsible for abduction of the eye, is not innervated by the abducens nerve. Rather, in many cases, the lateral rectus is aberrantly innervated by the oculomotor nerve. Mutations in several genes, including CPA6, have been linked to Duane syndrome. The CPA6 gene is located within a genomic locus named DURS1, previously implicated in Duane syndrome. However, the causative gene at the DURS1 locus has not been definitively identified. The possibility that a mutant CPA6 might be responsible for axon guidance defects leading to Duane syndrome prompted us to investigate the role of CPA6 in the zebrafish. This model organism enables the easy determination of developmental gene expression patterns and quantitative analysis of eye movement following perturbation of gene expression. Here we describe the cloning of Nilotinib zebrafish CPA6, its enzymatic activity and mRNA distribution during embryonic development. Although the expression pattern of CPA6 is consistent with a role in Duane syndrome, behavioral data show no effect on eye movement upon CPA6 knockdown. We propose that CPA6 may be involved in the etiology of Duane syndrome through expression in a relevant chondrogenic condensation, but dysfunction of additional genes and/or evolutionary modification of axon guidance is required for the manifestation of a phenotype. The expression pattern seen for zebrafish CPA6 is not shared by any of the other four CPA-like enzymes found in the zebrafish. Both CPA1 and CPA2 are detected in the pancreas after 3 dpf, consistent with their known roles as pancreatic digestive enzymes. Although no CPA3 was identified by sequence homology in the zebrafish, CPA5 appears to take on this role, with expression found in a population of cells recently identified as mast cells. The weak expression of CPA2 and CPA4 detected by RT-PCR early in development, and of CPA4 and CPA5 detected by in situ hybridization in the pancreas later in development, is consistent with previous reports of weak embryonic expression of human orthologs. For the most part, each CPA gene exhibits distributions consistent with its mammalian ortholog, yet distinct from that of CPA6. Of particular interest to this study was the conserved expression of CPA6 posterior to the eye, consistent with a role for CPA6 in the etiology of Duane syndrome. Although previous observations suggested that CPA6 was expressed in lateral rectus muscle precursor cells, it is difficult to distinguish these cells from developing chondrogenic tissue without markers. Based on the careful analysis of CPA6 distribution in zebrafish, it does not appear that CPA6 is expressed in lateral rectus muscle precursor cells or in any other myogenic cell population. Rather, the condensation near the eye in which zebrafish CPA6 is found is consistent with developing chondrogenic tissue. CPA6 is found in bony/chondrogenic tissues in the mouse also. In teleost fish, the chondrogenic tissue found next to the lateral rectus muscle subsequently forms the walls of a compartment known as the myodome.