The enzyme-linked immunospot (ELISPOT), a method for quantifying specific and total antibody-secreting cells, was used for the diagnosis of human immunodeficiency virus (HIV) infection in a prospectively followed cohort of infants born to HIV-infected women. From July 1, 1987, to June 1, 1990, 127 infants with known HIV infection status were studied. Seventeen of 22 HIV-infected infants had specific HIV-specific antibody-secreting cells (ASC). Among the infected infants rates of ASC positivity increased during the first year of life, from 25% in the first 5 days of life to 78% after 6 months. Two of the five ASC-negative infected infants were further characterized as hypo- or dysgammaglobulinemic by an adjunct ELISPOT assay for total immunoglobulin-secreting cells. Excluding hypo- or dysgammaglobulinemic infants from the analysis, the rate of ASC positivity among infected infants was 85% (17 of 20) after the age of 6 months. None of the 95 uninfected infants had a positive ELISPOT assay, including 55 who were tested in the first 3 months of life. Thus in this series the specificity was 100%. ELISPOT methodology can be a useful technique for the diagnosis of HIV infection in infants of HIV-seropositive mothers.