Guanylate cyclase-activating protein (GCAP) is a novel Ca(2+)-binding protein that stimulates synthesis of cGMP in photoreceptors. Molecular cloning of human and mouse GCAP cDNA revealed that the known mammalian GCAPs are more than 90% similar, consist of 201-205 amino acids, and contain three identically conserved EF hand Ca2+ binding sites. The sequence homology with recoverin, a related photoreceptor Ca(2+)-binding protein, is less than 35%. In situ hybridization in primate retinas shows that the GCAP gene is expressed exclusively in photoreceptor inner segments. To investigate the GCAP gene structure, we probed 10 eucaryotic genomic DNAs with a bovine GCAP cDNA under stringent conditions. The results demonstrate that the GCAP gene has been well conserved during evolution of vertebrate species and that each gene is most likely present as a single copy. By genomic cloning, polymerase chain reaction, mapping, and direct sequencing, we show that the human GCAP gene spans approximately 6 kilobases of genomic DNA, and consists of four exons (> 250, 146, 94, and 800 base pairs) separated by three introns (4.5 kilobases, 370 base pairs, and 347 base pairs). Using human/hamster hybrid panels and fluorescent in situ hybridization, the GCAP gene was localized to the short arm of chromosome 6 (p21.1).