Early diagnosis of infection with human immunodeficiency virus type 1 (HIV- 1) in young infants is essential to decisions on their medical and social care. Whereas studies have suggested that polymerase chain reaction (PCR) is a sensitive and timely method of diagnosing HIV infection in children, these evaluations have been limited by the number of specimens studied. Recently, Roche Molecular Systems developed a complete HIV-1 DNA PCR testing kit (from specimen preparation to detection). In this study, use of this PCR test kit was evaluated for the detection of HIV infection in infants of seropositive mothers who were enrolled in the longitudinal, multicenter Women and Infants' Transmission Study. A total of 1209 blood specimens from 483 infants were tested and analyzed. The overall sensitivity and specificity of a single PCR test in determining HIV infection status in infants more than 1 but less than 36 months of age were 95% and 97%, respectively. For infected infants 1 to 6 months of age the sensitivity of the DNA-PCR test was 90% to 100%. In a direct comparison with coculture, the Roche DNA-PCR test was significantly more sensitive than coculture in the detection of HIV-1 in infected infants and was equivalent to coculture for the diagnosis of HIV in infants when a standardized algorithm was used to define infection status.