Since acid treatment of serum is known to disrupt immune complexes, the diagnostic utility of the p24 antigen assay was examined after acid treatment of 345 serum samples from 158 children born to women infected with human immunodeficiency virus (HIV). Although the p24 antigen assay after acid treatment was negative in 9 HIV-1-infected children < 1 week old, antigen was detectable at high levels in all 30 samples obtained from infected children 1-9 months old. Overall, antigen was positive in 145 (sensitivity 89.5%) of 162 samples from 47 HIV-1-infected children > or = 1 month old. In contrast, the sensitivity of the p24 antigen assay without acid dissociation was only 18% (P < .001). Among the 76 uninfected children, 132 (specificity 99.2%) of 133 specimens were p24 antigen-negative after acid dissociation. These results demonstrate that acid treatment of serum markedly improves the sensitivity and predictive value of the p24 antigen assay for diagnosis of perinatally acquired HIV-1 infection in children 1 month of age or older.