Selective effects of in vitro azathioprine (AZ) on subpopulations of human lymphocytes involved in the pokeweed mitogen (PWM)-induced plaque forming cell (PFC) response against sheep red blood cells (SRBC) have been described. First, it was found that the responding B cell itself was very sensitive to AZ as concentrations as low as 0.01 µg/ml markedly suppressed the PWM-induced PFC responses of unfractionated peripheral blood mononuclear (MN) cell suspensions. The fact that AZ suppresses the PFC response of B cells only when added at the beginning of the cultures suggests that the drug interferes with some early events involved in B cell triggering by PWM. Furthermore, it was demonstrated that higher concentrations of AZ, i.e., 1 and 10 µg/ml selectively eliminated certain regulatory subsets of cells known to act as suppressors of this polyclonally induced PFC response. Preincubation of a) T cell-enriched (TCE) suspensions of cells, b) concanavalin A (Con A)-generated suppressor cells, and c) naturally occurring suppressor cells from nonresponder individuals with 1 and 10 µg/ml of AZ before co-culturing them with fresh MN cells in the presence of PWM caused a differential suppression of suppressor cell function and a selection for a pure helper effect with a resulting increase in B cell responses. Finally, regulatory helper cells were quite resistant to the in vitro effects of this drug as they still expressed their helper activity after pretreatment with 10 µg/ml AZ. Thus, this study demonstrated that B cells are highly sensitive, suppressor cells are less sensitive, and helper T cells are quite resistant to the in vitro effects of AZ.