Sera from prospective transplant patients are usually screened for HLA antibodies prior to transplantation, but presently available tests do not permit quantification of the humoral alloantigen directed response. We adapted a culture system for isolated human B-lymphocytes to assay the secretion of HLA-antibodies on a single cell basis. B-cell supernatants were screened for HLA antibodies by complement dependent cytotoxicity. The assay assigns precursor frequencies for HLA-alloantibody secreting B-lymphocytes (BCPFs), and simultaneously allows for dissection of the humoral alloantigen directed response into its monoclonal components. The lymphocytes of 15 HLA-seropositive multiparous women that were used to validate the assay, were found to contain HLA-BCPFs ranging from 0 to 123 per 10(6) B-lymphocytes (mean: 43 +/- 45 per 10(6) B-lymphocytes). The HLA-specificities of antibodies in the B-cell supernatants were in agreement with serum specificities. Genuine HLA reactivity of B-cell supernatants was confirmed using an ELISA with purified HLA class I antigens. When applied to lymphocytes of patients on transplant waiting lists, the present assay may enable the unraveling of serum specificities in their components, thus supplementing HLA antibody serum screening data.