An early and accurate diagnosis of HIV infection is needed in the offspring of seropositive mothers. To this end, we have used two techniques for the direct detection of HIV in 12 newborns tested within 2 weeks after birth and 12 children. HIV isolation was carried out in lymphocyte cocultures and compared with detection of DNA and RNA sequences by molecular amplification using the polymerase chain reaction (PCR). In lymphocyte cocultures, HIV was isolated in 8 of 24 cases (33%), including 3 newborns, 3 symptomatic children, and 2 asymptomatic ones. HIV DNA was detected by PCR in twice as many cases, i.e., in 16/24 cases (66%), including 7/12 newborns, 4/4 symptomatic children, and 5/8 asymptomatic ones, 2 of whom became seronegative, HIV RNA was detected in 10 of 16 cases (60%) with detectable HIV DNA, including all of the cases who had a positive HIV isolation. Only children with clinical or biological signs of HIV infection were positive for HIV RNA. Furthermore, signs of HIV infection appeared within 6 months in three of the four newborns who were positive for HIV RNA at birth. These results indicate that HIV DNA detection by PCR is far more sensitive than HIV isolation in culture for the early diagnosis of HIV infection in offspring of seropositive mothers. HIV RNA detection appears to be a useful prognostic marker since it does correlate with disease progression and may serve as a clue for HIV replication in vivo.