The O and R antigenic variants of the Plasmodium falciparum Palo Alto strain present differences in the morphology of the infected red blood cell membrane, in their adhesion properties, surface immunofluorescence, and agglutination specificities and importantly, induce a variant-specific protection after a primary infection in Saimiri sciureus monkeys. To identify potential targets of variant-specific immunity, we have compared the antigenic makeup of both variants by immunoblot. O-specific monkey sera generated similar profiles on both parasite types, while R-specific sera showed a consistent difference on a high-molecular-mass undefined antigen. Distinct antibody specificities were eluted from the surface of O- or R-infected erythrocytes, generating variant-specific agglutination, surface immunofluorescence, and immunoblot profiles. An antiserum raised to Pf60.1, predicted to cross-react with the cytoplasmic domain of PfEMP1, reacted with specific, SDS-soluble antigens in both variants. Antigens associated with the membrane of the infected red blood cells were further investigated using several specific antisera. The 85-kDa HRP1 gene product was more abundant in O than in R parasites, while the reverse was observed for the PfEMP3 protein. These data indicate that O and R parasites differ in the expression of several antigens associated with the membrane of the infected red blood cell.