The enzyme choline acetyltransferase [EC 2.3.1.6] (ChAT) synthesizes the neurotransmitter acetylcholine that plays a key morphogenic role in vertebrate retina development. As the embryonic avian retina is particularly useful for morphogenetic studies, we cloned the complete coding region of chicken ChAT cDNA. At the deduced amino acid level, chicken ChAT is approximately 76% identical to mammalian ChAT proteins. We also report here the cloning of the complete 5' end of the complex cholinergic locus. This locus contains both the ChAT gene and the nested intronless gene for the vesicular acetylcholine transporter (VAChT). The genomic organization of the 5' end of the chicken cholinergic locus is similar to that reported in other vertebrate species. A 5.7 kb mRNA corresponding to the ChAT message was detected in both embryonic retina and post-hatch brain. An analysis of the ChAT mRNA in embryonic chick retina shows that the message can be detected by E6 and its level increased during early retinal development. Vertebrate ChAT mRNAs can contain one or more of three non-coding exons, M, N or R and by RT-PCR we demonstrate, at least, a chicken ChAT mRNA containing exon M.