A simple method to distinguish between simian immunodeficiency virus (SIV) isolates of experimentally infected rhesus macaques is reported. Peripheral blood mononuclear cells (PBMC) were prepared from a rhesus macaque infected with SIVStM isolated originally from a stump-tailed macaque, or from a rhesus monkey infected with SIVSM from a sooty mangabey monkey. PBMC were cocultivated with CEM × 174 cells and a region of the SIV envelope (env) gene was amplified by the polymerase chain reaction (PCR) from cDNA of infected cocultivation cells. Restriction enzyme digestion analysis of the PCR products enabled SIVStM and SIVSM to be differentiated from each other, and from a molecular clone of SIVMAC, SIVMAC239, originally isolated from an infected rhesus macaque. Furthermore, when SIVSM and SIVStM were introduced into the same animal, restriction enzyme analysis of the PCR product amplified from cocultivation cells of this rhesus macaque suggested that the animal was superinfected.