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To validate a method for salivary testing for HIV infection in children older than 12 months.Oral fluid samples were collected via sponge foam swabs from children born to HIV-positive mothers and were tested for antibodies to HIV-1 and HIV-2 with an IgG antibody capture enzyme-linked immunosorbent assay and a modified Western blot for confirmation. In each child serum antibody status was the standard used to validate the salivary antibody test.We obtained 331 oral fluid samples from children born to HIV-positive mothers. The specificity and sensitivity of salivary testing compared with results on sera were both 100% (297 of 297 (95% confidence interval 98.8 to 100%) and 34 of 34 (95% confidence interval 89.7 to 100%), respectively). Compliance in the study population increased from 91% to 97% when mothers were offered the opportunity to provide oral fluid from their children instead of blood specimens.Salivary testing provides an accurate and acceptable noninvasive method for assessing the HIV infection status of children born to infected mothers by using IgG antibody capture enzyme-linked immunosorbent assay alone with a strategy of duplicate retesting of reactive specimens.