Using normal human lymphocytes isolated by sedimentation and cotton column adherence, we have developed a reliable assay of immunosuppression of PHA-induced blastogenesis by serum from selected melanoma patients. These lymphocyte cultures contained both responder cells (subpopulation x) and regulator cells (subpopulation y). Lymphocytes isolated by gradient centrifugation on sodium metrizoate-Ficoll contained responder cells (x) but no regulator cells (y). Cultures of lymphocytes isolated by this method were stimulated by PHA but were not suppressed by the addition of melanoma serum. When lymphocytes were isolated by a cotton column adherence/Lymphoprep centrifugation-double separation, subpopulations (x) and (y) were isolated. We have established that both subpopulations are necessary for suppression to occur, and that (y) operates as the regulator of (x). Finally, by manipulating B cell and T cell populations isolated by nylon column adherence or AET rosette separation, we have determined that the regulator ability of subpopulation (y) is the result of B cell activation of suppressor T cells.