We have isolated and characterised the gene encoding the chicken axonal cell adhesion molecule axonin-1. This gene comprises 23 exons distributed over approximately 40 kb. Each of the six immunoglobulin-like domains and the four fibronectin-type-III-like domains of axonin-1 is encoded by two exons. The introns between two domains are exclusively phase I. Their exon/intron borders correspond to the domain borders of the protein, suggesting that the gene of axonin-1 had been generated by exon shuffling. Three transcripts with a length of 4.3 kb, 5 kb, and 8 kb are found, and we provide evidence that they result from alternative use of polyadenylation signals. In situ hybridization revealed co-localisation of these transcripts in time and space in the developing chicken retina. Several identical transcription initiation sites were found in retina, brain, and cerebellum by RNase protection assay and anchored polymerase chain reaction. By transfection of HeLa cells, rat PC-12 phaeochromocytoma cells, and chicken embryonic fibroblasts with serially truncated segments of the 5'-flanking region linked to a luciferase reporter gene, we have found that the sequence from -91 to +56 relative to the transcription initiation site is sufficient to promote efficient gene expression. Tissue-specific expression of the axonin-1 gene seems to be regulated in part by sequences more than 1 kb upstream of the transcription initiation site. As revealed by computer analysis, the sequence immediately upstream of exon 1 contains an AP-2 binding site, a tumor phorbol-ester-responsive element, and a homeodomain protein binding site, but no canonical TATA box. A second AP-2 binding site and a homeodomain protein binding site are located within exon 1.