A plasma HIV-1 RNA amplification assay (RNA assay), a quantitative peripheral blood mononuclear cell (PBMC) microculture (culture), and a PBMC HIV-1 DNA amplification assay (DNA assay) were compared for diagnosis of HIV-1 infection in infants receiving zidovudine in Pediatric AIDS Clinical Trials Group protocol 185; assays were performed for all 24 infected and 100 uninfected infants. HIV-1 infection was defined as >or=2 positive cultures or positive antibody to HIV-1 at >or=18 months. Cultures were performed at birth and 6 and 24 weeks of age; DNA and RNA assays were performed on cryopreserved specimens. The sensitivity of culture and DNA and RNA assays at birth was 20.8%, 10.5%, and 26.7%, respectively. At older ages, sensitivity typically exceeded 80%, remaining highest for the RNA assay (>85%). Assay specificity was >99%. Positive predictive values exceeded 93% for each assay at each age; negative predictive values were highest (>90%) for the RNA assay. At birth (P < 0.005) and age 6 weeks (P < 0.001), a significantly larger proportion of infected infants were identified by means of the RNA assay than by the other assays. The diagnostic performance of the RNA assay matched or exceeded that of culture and the DNA assay. Given that RNA assays require less blood volume and yield rapid results, our study adds to existing data suggesting that RNA assays may be used for early diagnosis of HIV-1 infection in infants.