The cDNA coding for mouse and human ras p21 GTPase-activating protein (GAP) was isolated; the deduced amino acid sequences share over 96% homology with that previously determined for bovine brain GAP. Both the mouse and human GAP cDNAs were used as probes for the chromosomal localization of this gene. The locus designations for the gene encoding GAP in human and mouse are RASA and Rasa (for ras-activating protein), respectively. By somatic cell hybrid analysis and in situ chromosomal hybridization, we have assigned the RASA gene to human chromosome band 5q13.3. In addition, with somatic cell genetics and linkage analysis in recombinant inbred mouse strains, the murine Rasa gene was localized to the distal end of mouse chromosome 13. These assignments place the gene encoding GAP in a known conserved syntenic region.