Serial blood cultures over the first 6 months of life in 310 infants with vertical exposure to human immunodeficiency virus (HIV) in the Women and Infants Transmission Study were analyzed to determine their value for early diagnosis of HIV infection. Cultures were done at 0–7 days and 1, 2, 4, and 6 months of age: 55 infants were infected. Blood culture sensitivity in infected children was 24% (7/29) during the first week of life and 85%, 91%, 82%, and 88%, respectively, at 1, 2, 4, and 6 months. The sensitivity, specificity, and positive and negative predictive values of a single culture between 1 and 6 months of age were, respectively, 86.9%, 99.6%, 97.9%, and 97.5%. Two negative cultures between 1 and 6 months of age defined an uninfected infant with a specificity of 99.2%-100.0%. Blood culture done between 1 and 6 months of age in children of HIV-positive mothers is a sensitive and specific test for HIV infection, with high positive and negative predictive values.