In vitro treatment with 2 micrograms/2 X 10(6) cells melphalan (L-PAM: L-phenylalanine mustard) significantly decreased the total number of T lymphocytes from peripheral blood (PBL) of healthy human donors and of the OKT4 population (precursor suppressor/helper/inducer) T cells as defined by monoclonal antibodies OKT3 and OKT4, respectively. No changes in the OKT+8 lymphocyte population (cytotoxic/mature suppressor cells) were observed following the same treatment. Preincubation of PBL with L-PAM at concentrations that do not affect the rate of DNA synthesis in PHA-stimulated lymphocytes inhibited the generation of T suppressor lymphocytes by ConA, as shown by their effect on PHA stimulation. Treatment of allogeneic PBL with L-PAM had no effect on mature suppressor T cells already induced by ConA, as shown by incubation of PBL with L-PAM after incubation with ConA.