Because western blotting occasionally causes cross-reactions between human immunodeficiency virus (HIV)-1 and HIV-2, it is difficult to distinguish a coinfection status from a false-positive result. Therefore, we developed a qualitative real-time PCR assay to detect HIV-1 and HIV-2 RNA that can be performed in parallel. Viral RNA extracted from 500 μl of plasma was examined using real-time PCR with minor groove binder probes. Bovine leukemia virus was used as an internal standard. The sensitivity was determined by probit regression analysis using the World Health Organization international standards for HIV-1 and HIV-2. The lower detection limits at a 95% hit rate were 54 IU/ml for HIV-1 and 5.0 IU/ml for HIV-2, which were lower than any HIV-2 assays reported previously. HIV-1 RNA was detected in 51 of 52 HIV-1 seropositive plasma samples. HIV-2 RNA was detected in 7 of 10 HIV-2 seropositive plasma samples. Non-specific signals and cross reactivity between HIV-1 and HIV-2 were not observed in 100 HIV seronegative samples. The assay developed in this study is highly sensitive and specific for the detection of HIV-1 and HIV-2 RNA. The test is expected to be useful for the differential diagnosis of HIV-1 and HIV-2 infections.