Synapsin I is a neuron-specific phosphoprotein associated with the membranes of small synaptic vesicles. Its function is not entirely clear, but evidence points to a possible role in the regulation of neurotransmitter release. Its biosynthesis is under developmental control. Assignment of the human synapsin I gene to the X chromosome at band Xp11 was accomplished by in situ hybridization, using a rat cDNA probe. Southern blot analysis of DNAs from a panel of human-Chinese hamster somatic cell hybrids with defined regions of the human X chromosome confirmed the in situ mapping data. The mouse synapsin I gene was assigned to the X chromosome, proximal to band XD, by Southern blot analysis of Chinese hamster-mouse somatic cell hybrids with normal or rearranged mouse X chromosomes. In situ chromosomal hybridization experiments localized the mouse synapsin I gene more precisely to bands XA1----A4. These results add to the comparative gene map of mammalian species and support certain hypotheses regarding the evolutionary relationship between human and mouse X chromosomes. We hypothesize that the synapsin I gene could be mutated in human X-linked disorders with primary neuronal degeneration, such as the Rett syndrome.