Supernatants of fresh human MNL stimulated with concanavalin A (Con A) generally contain little or no lymphocyte-derived mitogenic factor (LMF or Interleukin-2). However, preincubation of MNL in culture medium for 2 to 5 days before stimulation with Con A rendered them capable of producing increasing LMF. The production of LMF by reincubated MNL was inhibited by readdition of autologous fresh MNL or E-rosetting (T) lymphocytes. The producing cells were T lymphocytes; however, the production of LMF was monocyte dependent. LMF eluted from a Sephadex G150 column in 2 peaks at 12 to 27,000 and 50 to 70,000 daltons. Both species of LMF were mitogenic for mouse (C3H/HeJ) thymocytes and for human T and B cells. LMF production was not affected by x-irradiation (less than or equal to 2500 R) before preincubation; it was, however, abolished by the addition of 10(-7) M hydrocortisone during the preincubation period. LMF production by fresh MNL could be moderately enhanced by the addition of cimetidine, a drug that is known to interfere with histamine H2 receptors on suppressor T cells. Presumably, preincubation of MNL reduces the activity of Con A-induced suppressor cells. We conclude that human peripheral blood contains suppressor T cells that when activated by Con A regulate the production of LMF by other T cells in response to Con A.