The objective of this study was to assess the frequency of detection of HIV-1 RNA in plasma of neonates born to HIV-1-seropositive mothers and to determine the diagnostic value of this method in the neonatal period. The study involved 96 infants among those enrolled in the French National Prospective Study. HIV-1 RNA was detected in the first 10 days of life by nucleic acid sequence-based amplification (NASBA) in 12 of 48 plasma samples of infected infants and in 39 of 39 of the second samples taken before the age of 3 months. On the same samples, peripheral blood mononuclear cell (PBMC) DNA polymerase chain reaction (PCR) or viral culture that had been routinely performed were found to be positive in 11 of 48 samples taken in the first 10 days of life and 39 of 39 second samples. For the noninfected infants, HIV-1 RNA was never detected in the 48 samples taken in the first 10 days of life and was detected in one of the 48 samples taken before the age of 3 months. HIV-1 RNA detection in plasma by NASBA has sensitivity and specificity equal to those of DNA PCR and culture on PBMC for the diagnosis of infection in infants with the clade B virus. This standardized method gives rapid results on a small volume of plasma and seems well suited for diagnosis on a large scale.