We report the production of a lymphoblastoid human B cell line, which secretes monoclonal antibody with HLA-DRw53-like specificity. The immune lymphocytes were obtained from a patient who had been undergoing immunotherapy at Memorial Hospital with allogeneic cultured melanoma cells. The antibody producing cell line was established by transformation of immune B cells with Epstein-Barr virus and stabilized by serial selection of antibody-positive cell clusters. The cell line (R39.14) has been producing a cytotoxic antibody of IgM, kappa type during nearly two years of continuous culture. The specificity pattern was determined on a panel of HLA-DR-typed lymphoblastoid cell lines, and resembled the HLA-DRw53 specificity closely. In studies of informative families the R39.14 specificity segregated with DRw53-bearing haplotypes, which included DR-4, 7 and DRw9 antigens. The R39.14 specificity was found to be expressed in similar quantities on all antibody positive homozygous cell lines tested. Blocking experiments with monoclonal class II specific antibodies revealed R39.14 to reside on a DR molecule in close association with the determinant detected by the 109d6 mouse monoclonal antibody.