The Women and Infants Transmission Study (WITS) has established virologic definitions of human immunodeficiency virus (HIV)-infected and uninfected children that have been widely used but never formally compared with serologic definitions of infection. Data from the offspring of HIV-infected women in the WITS with frequent HIV cultures during the first year of life and with HIV serology at 18 and/or 24 months of age were analyzed. Seventy-seven infants were HIV-infected and 430 uninfected by both virologic and serologic criteria. Thirteen were virologically infected (> or = 2 positive cultures) but either seronegative or serologically indeterminate. All but 1 of these had clinical HIV disease at the time of analysis. In this pediatric cohort, children defined as infected by virologic criteria often (13/90) had negative or indeterminate serology despite symptoms of HIV disease. Results suggest that serology at 18-24 months has high specificity but poor sensitivity. It should not be considered the reference standard in identifying HIV infection in perinatally exposed children.