SNAP-25 (synaptosomal associated protein of 25 kDa) is a neuronal-specific but non-uniformly expressed protein. Expression is correlated to the time of synaptogenesis and SNAP-25 protein is found predominantly in presynaptic fields. Here we report the cloning and characterization of the SNAP-25 chicken gene. The gene spans more than 65 kb of genomic DNA and is interrupted into nine different exons. Identification of the 5'-end of the mRNA showed that transcriptional initiation is heterogeneous, although a major transcriptional start site is present 30 bp downstream of a putative TATA-box. Analysis of the exon-intron organization revealed that exon 5 exists in two homologous but distinct versions separated by 140 base-pairs. Both exons 5 are expressed as mRNA, indicating that alternative splicing regulates the expression of two isoforms, a and b, of the SNAP-25 protein. The alternative splicing results in a difference of nine amino acid residues between the proteins in a domain demonstrated to be subject to palmitoylation. This implies that the two isoforms differ in their capacity or efficiency to be modified by fatty acylation, suggesting divergent abilities to interact with neuronal membranes.