Among the small GTPases of the Ras family, Rap proteins exhibit the highest homology with p21Ras. The four Rap proteins so far identified constitute two subgroups, comprising the Rap1(A,B) and the Rap2(A,B) proteins. The intracellular location of Rap1A, Rap1B and Rap2A proteins was investigated in mammalian cells by confocal immunofluorescence microscopy. Using a specific anti-Rap1 affinity-purified antibody, both Rap1A and Rap1B proteins were localized to late endocytic compartments (late endosomes/lysosomes) in fibroblasts. The localization of the Rap1A and B proteins transiently overexpressed with the vaccinia T7 system was identical to that observed for endogenous Rap1 proteins. In contrast, epitope-tagged Rap2A protein colocalized with several markers of the Golgi complex, thus indicating that its site of function was distinct from that of Rap1A. In addition, morphological and subcellular fractionation studies provided evidence for the association of Rap1 proteins with phagosomes displaying biochemical features of late endocytic structures in J774 macrophages. Thus, the localization of Rap1A and Rap1B implicates their involvement in late endocytic/phagocytic processes.