More than one-half of the children born to women with human immunodeficiency virus (HIV) infection are not infected with HIV. Most of these children, although born antibody-positive, lose maternal antibody and remain asymptomatic. However, it has been unclear how many antibody-negative children of HIV-infected women may truly be infected despite the loss of passively acquired maternal antibody. One hundred nine children who lost maternal antibody after birth to HIV-infected women recruited in four United States maternal HIV transmission studies were examined for HIV infection. Polymerase chain reaction (PCR) was used to determine whether children had HIV proviral DNA in peripheral blood mononuclear cells. A total of 268 samples from 109 children were tested. Clinical status and other laboratory findings were also evaluated. The median age at last follow-up was 25 months (range, 12 to 48 months). One hundred seven (98%) children were negative by PCR on all samples tested. None (95% confidence interval, 0.0 to 1.9%) of 109 children had a repeatedly positive PCR. Two children had single positive PCR results that could not be confirmed on subsequent testing. No other laboratory or clinical findings supported HIV infection in either of these children. The loss of HIV antibody in an asymptomatic child born to an HIV-infected woman strongly suggests that the child is not infected with HIV. Single positive PCR results, particularly in the absence of other clinical or laboratory evidence of HIV infection, should not be used alone to diagnose HIV infection.