To approximate the contributions of in utero, intrapartum, and postnatal transmission of human immunodeficiency virus type-1 (HIV-1) and to evaluate polymerase chain reaction (PCR) as a diagnostic tool for pediatric HIV infection, blood was collected at birth (cord blood), and at 3, 6-12, and 13-24 months in 218 children born to HIV-1-seropositive mothers in Kigali, Rwanda. Proviral DNA was detected by a double PCR using two sets of three primers (gag, pol, and env). Pediatric HIV-1 infection was defined according to serological and clinical criteria. The probability of having a positive PCR at a given time was calculated by a nonparametric method. Among children with unequivocal evidence of infection (n = 47), it was 30.5% on cord blood and 80.6% at 3 months. Thus, in children born to HIV-1-infected mothers, the estimated rate of transmission in the late postnatal period is 4.9%, and the rate of transmission in the intrapartum plus postnatal periods is 17.6%. Among 117 HIV-1-uninfected children born to HIV-1-infected mothers, six (5%) had a false-positive PCR on cord blood. These results should be taken into account in designing intervention trials aimed at reducing mother-to-child transmission of HIV-1.