Quantitative HIV RNA testing was used for diagnosis in 156 HIV-exposed non-breast-fed infants at less than 6 months of age (54 infected, 102 uninfected) enrolled in the Perinatal AIDS Collaborative Transmission Study. Sensitivity was 29% in the first week, 79% at 8 to 28 days of age, and >90% at 29 days of age and thereafter; specificity was 100% in all periods, except at 29 to 60 days of age, when specificity was 93%. Neither sensitivity nor specificity was significantly affected by maternal or infant zidovudine (ZDV) treatment, even though infant viral loads were lower during the first 6 weeks in infants who received perinatal ZDV prophylaxis ( p=.005). Paired analysis of DNA and RNA measurements revealed no advantage for either test. Quantitative RNA testing can be used for diagnosis in HIV-exposed infants, recognizing the chance for a false-positive test result. It may be most useful as a confirmatory test in infants with another positive diagnostic test result.