Synaptophysin is an integral membrane protein of small synaptic vesicles in brain and endocrine cells. We have determined the structure and organization of the human synaptophysin gene and have established the chromosome localizations in man and mouse. Analysis of a cosmid clone containing the human synaptophysin gene (SYP) revealed seven exons distributed over approximately 20 kb, when compared with the previously published cDNA sequence. The exon-intron boundaries have been identified and do not correlate with functional domains. One intron interrupts the 3' untranslated region. Chromosomal localization of the human and murine genes for synaptophysin established the human SYP locus on the X chromosome in subbands Xp11.22-p11.23 and the mouse synaptophysin gene locus (Syp) on the X chromosome in region A-D. In addition, an Eco0109 RFLP has been identified and used in genetic mapping of the human SYP locus and supports the order TIMP-SYP-DXS14 within a span of approximately 4-7 centimorgans.