Incubation of [3H]palmitic acid, ATP, and CoA with inside-out membrane vesicles prepared from human or other mammalian red cells resulted in nearly exclusive 3H-palmitoylation of the Mr = 32,000 Rh polypeptides. [3H]Palmitic, [3H]myristic, and [3H]oleic acids were comparably esterified onto Rh polypeptides in inside-out membrane vesicles in the presence of ATP and CoA, although [3H]palmitic acid was preferentially incorporated by intact human red cells. Experiments using sulfhydryl reagents or tryptic digestions suggested that multiple sulfhydryl groups on the Rh polypeptides located near the cytoplasmic leaflet of the lipid bilayer were 3H-palmitoylated; the exofacial sulfhydryl group essential for Rh antigenic reactivity was not 3H-palmitoylated. Transfer of fatty acid from [14C]palmitoyl-CoA to sites on the Rh polypeptides occurred even after previous incubation of inside-out membrane vesicles at 95 degrees C or after solubilization of inside-out membrane vesicles in Triton X-100. Hydrodynamic analyses of Triton X-100-solubilized membranes surprisingly demonstrated that 3H-palmitoylated Rh polypeptides behaved as a protein of apparent Mr = 170,000. These in vitro studies suggest that palmitoylation of Rh polypeptides occurs within a macromolecular complex by a highly selective but possibly nonenzymatic mechanism.