We report on an investigation designed to compare the polymerase chain reaction (PCR) with culture and p24 measurement for the diagnosis of human immunodeficiency virus (HIV) infection in infants and children. Forty-five children born of mothers with antibodies to HIV type 1 were studied; P24 antigen was measured in plasma, and HIV-1 proviral DNA was sought in peripheral blood mononuclear cells after amplification by PCR. In 26 cases, blood specimens were cultured for HIV; in all but two instances cultures were established at the same time that the PCR test was performed. Primer pairs in three regions of the proviral genome were used for the PCR test. There was good agreement between the results obtained from PCR tests and from cultures; of 24 children in whom both tests were done at the same time, 10 had positive results on both the culture and the PCR test, 1 had positive results on the PCR test but negative culture results, and 13 had negative results on both tests (concordance 96%). Measurement of p24 antigen in plasma was, in contrast, an insensitive marker of infection: 6 of 12 infants with positive cultures had positive p24 test results, and 8 of 18 infants had positive PCR test results. Sixteen children with subsequent seronegativity for HIV-1 had negative PCR results. This study provides further evidence that the PCR test is a valid alternative to viral culture for the diagnosis of pediatric HIV infection.