In vitro treatment with melphalan (l-PAM, l-phenylalanine mustard), 2 μg 2 × 106 cells, significantly decreased the total number of E-rosette-positive (E+) T lymphocytes from peripheral blood (PBL) of healthy human donors as well as those of the OKT4 (precursor suppressor/helper/inducer T cells) and OKT17 populations (suppressor cells within the OKT4 subset). The OKT8 population (cytotoxic/mature suppressor cells) was not affected by a similar l-PAM treatment. The sensitivity of concanavalin A (Con A)-activated E+ T-cell populations to subsequent l-PAM treatment in vitro was different from that of Con A-untreated T cells: Thus, l-PAM treatment did not affect the expression of OKT3 and OKT4 antigens, increased the percentage of OKT17 cells, and inhibited the experssion of OKT8 antigen. Depletion of OKT8 from Con A-activated E+ T cells (OKT4+-OKT8--OKT17+) did not affect their suppressive activity on PHA stimulation in l-PAM-treated as well as untreated cells. Further depletion of OKT17+ cells from the OKT4+-OKT8--OKT17+ subset (OKT4+-OKT8--OKT17-) abolished the suppressive effect on PHA stimulation. Suppressive activity of the OKT4+-OKT8--OKT17- subset was again evident after treatment of this population with l-PAM. The results obtained indicate that the sensitivity to l-PAM treatment of various T-cell phenotypes is changed by Con A activation and that after depletion of specific T suppressor cells l-PAM seems to affect the immunoregulatory circuit within the Con A-activated OKT4 subset.