Diverse GTPase-activating proteins (GAPs) for the p21rho subfamily were detected by a novel overlay assay (Manser, E., Leung, T., Monfries, C., Teo, M., Hall, C., and Lim, L. (1992) J. Biol. Chem. 267, 16025-16028), with some GAPs being tissue-specific. Using a PCR strategy exploiting conserved regions common to rho/rac-GAPs, we have isolated a rat testis cDNA encoding a 34-kDa rac-GAP termed beta-chimaerin, as it was highly related to n-chimaerin, containing both a GAP domain (77% identity) and the phorbol ester-binding region (93% identity). beta-Chimaerin mRNA is expressed exclusively in the testis at the onset of sexual maturation. In situ hybridization and cell fractionation analyses show beta-chimaerin mRNA expression to be stage-specific, paralleling acrosomal assembly at the late stage of spermatogenesis. A corresponding testis-specific 30-kDa rac-GAP was detected. The testis-specific and stage-dependent expression of this new member of the chimaerin family offer an alternative model system for investigating the functional role of this class of p21 GAPs, particularly in relation to cytoskeletal reorganization.