Accurate determination of plasma human immunodeficiency virus type 1 (HIV-1) RNA levels is critical for the effective management of HIV-1 disease. The AMPLICOR HIV-1 MONITOR Test, a reverse transcription-PCR-based test for quantification of HIV-1 RNA in plasma, was developed when little sequence information on HIV-1 isolates from outside North America was available. It has since become apparent that many non-subtype B isolates, particularly subtypes A and E, are detected inefficiently by the test. We describe here the AMPLICOR HIV-1 MONITOR Test, version 1.5, an upgraded test developed to minimize subtype-related variation. We also developed a panel of HIV-1 standards containing 30 HIV-1 isolates of subtypes A through G. The virus particle concentration of each cultured viral stock was standardized by electron microscopic virus particle counting. We used this panel to determine the performance of the original AMPLICOR HIV-1 MONITOR Test and version 1.5 of the test with HIV-1 subtypes A through G. The original test underestimated the concentration of HIV-1 subtype A, E, F, and G RNA by 10-fold or more, whereas version of the 1.5 test yielded equivalent quantification of HIV-1 RNA regardless of the subtype. In light of the increasing intermixing of HIV-1 subtypes worldwide, standardization of PCR-based tests against well-characterized viral isolates representing the full range of HIV-1 diversity will be essential for the continued utility of these important clinical management tools.