The gene for the small subunit of human calcium-activated neutral protease was isolated and sequenced. It is 11 kb long and comprises 11 exons. No TATA or CAT box was found upstream of the possible transcription initiation sites, but there are three so-called G-C box sequences and one G-C box-like sequence, which are usually found in "house-keeping" genes. The first exon (exon 1) contains only the 5'-noncoding sequence and exon 2 encodes the Gly-rich hydrophobic domain. Each of the four calcium-binding loop regions is encoded by one exon (exons 7-10). The intron breakpoints in the C-terminal calcium-binding domain (exons 4-11) completely coincide with those of the chicken large subunit gene. These findings suggest that the small and large subunits have evolved from the same ancestral calcium-binding protein and have retained the original gene organization.