Synaptobrevins 1 and 2 are small integral membrane proteins specific for synaptic vesicles in neurons. Two cosmid clones containing the human genes encoding synaptobrevins 1 and 2 (gene symbols SYB1 and SYB2, respectively) were isolated and characterized. The coding regions of the synaptobrevin genes are highly homologous to each other and are interrupted at identical positions by introns of different size and sequence. Each gene is organized into five exons whose boundaries correspond to those of the protein domains. Exon I contains part of the initiator methionine codon whereas exon II encodes the variable and immunogenic amino-terminal domain of the synaptobrevins. The third exon comprises the highly conserved central domain of the synaptobrevins, exon IV encodes most of the transmembrane region, and exon V contains the last residues of the transmembrane region and the small intravesicular carboxyl terminus. Comparisons of the synaptobrevin sequences in five species from Drosophila with man indicate a selective conservation of sequences adjacent to the synaptic vesicle surface, suggesting a function at the membrane-cystosol interface. The chromosomal localizations of the human and mouse SYB1 and SYB2 genes were determined using hybrid cell lines. SYB1 was localized to the short arm of human chromosome 12 and to mouse chromosome 6 whereas SYB2 was found on the distal portion of the short arm of human chromosome 17 and on mouse chromosome 11. A PstI restriction fragment length polymorphism was identified at the SYB2 locus.