A quantitative reverse transcriptase PCR assay with automated detection by nonradioactive hybridization was developed for the determination of human immunodeficiency virus (HIV) type 1 RNA levels. This assay is based on the use of an external standard curve with an internal standard. The accuracy of quantification was verified by comparison with reference commercial tests, the Chiron branched-DNA and Roche AMPLICOR HIV MONITOR assays. This assay was able to quantify viremia in patients with CD4 cell numbers below and above 500/mm3 and to quantify some HIV strains which could not be titrated by the MONITOR assay.