Previously described methods for measuring specific antibody production in vitro are complex and frequently require the addition of a non-specific mitogen and 2 or more separate steps including a lymphocyte culture and then an antibody assay. In this report we present further details regarding a 1-step, non-mitogen requiring assay for specific antibody synthesis combining microculture and enzyme-linked immunoassay techniques. Peripheral blood mononuclear cells from recently immunized subjects are cultured in antigen-coated plastic wells and the specific antibody produced and adhered to the solid-phase antigen is measured in an enzyme-linked immunoassay. The production of specific antibody in vitro did not occur in lymphocyte preparations that were frozen, thawed and incubated, nor did it occur in cultures containing puromycin. T cell separation and remixture experiments indicated that T cells provide significant augmentation under the conditions of this assay. It is apparent that the antibody measured was synthesized in vitro and that cell-cell interactions are operant. The assay may prove useful in the assessment of immune competence and in determining in vitro the effect of certain biologic response modifiers on antibody production.