rap1/Krev-1 is a p21ras-related GTP-binding protein that has been implicated in the reversion of the ras-transformed cell phenotype. We have identified a GTPase-activating protein (GAP) specific for rap in plasma membranes isolated from differentiated HL60 cells. The rap GAP activity remained quantitatively associated with the membrane following washes with buffered 1 M LiCl containing 20 mM EDTA but was solubilized with the detergents Nonidet P-40 and deoxycholate. On the basis of size-exclusion chromatography, the membrane-associated rap GAP (rap GAPm) appeared distinct from the rap GAP detected in the cytosolic fraction from HL60 cells. The molecular sizes of the membrane and cytosolic forms were estimated to be 36 and 54 A, respectively. rap GAPm was solubilized and purified to near homogeneity by successive column chromatographies in the presence of detergent. The rap GAPm activity corresponded to a single polypeptide that migrated with a molecular mass of approximately 88 kDa on SDS/polyacrylamide gels. The purified rap GAPm was inactive toward the GTP-bound forms of p21ras, rho, G25K, and rac-1 and did not stimulate dissociation of guanine nucleotide from rap.