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To determine the optimal time for a second HIV-1 nucleic acid amplification assay to detect late postnatal transmission of HIV-1 (first negative test at 4–8 weeks of age) in resource-limited settings.A longitudinal analysis of data from HIV Prevention Trial Network trial 024.Children born to HIV-1-infected mothers enrolled in the HIV Prevention Trial Network trial 024 were tested for HIV-1 infection at six intervals within the first year of life. Mothers and infants received nevirapine prophylaxis. We estimated the probability of being alive and having a positive test in each interval after 4–8 weeks and at 30 days after weaning, conditional on having acquired HIV during the late postnatal period. The interval with the highest probability was taken to be the optimal visit interval.A total of 1609 infants from HIV Prevention Trial Network trial 024 had at least one HIV-1 diagnostic test and were included in the analysis. We found that testing at 1 month after weaning or 12 months of age (whichever comes first) identified 81% of those infected during the late postnatal period (after 4–8 weeks) through breastfeeding. In total, 93% (95% confidence interval 89, 98) of all infected infants would be detected if tests were performed at these two time points.In resource-limited settings, HIV-1 PCR testing at 4–8 weeks followed by a second test at 1 month after weaning or at 1 year of age (whichever comes first), led to the identification of the vast majority of HIV-1-infected infants.