Murine peritoneal macrophages (M phi s), induced with stimuli such as thioglycollate, zymosan A, OK-432, bacille Calmette-Guérin (BCG), or live Mycobacterium intracellulare, showed varying levels of inhibitory activity against the concanavalin A (Con A) blastogenic response of splenic T cells. All test M phi s significantly inhibited the interleukin 2 (IL-2)-producing ability of T cells but this inhibition was not enough to explain the observed reduction in T cell Con A mitogenesis. In contrast, they markedly inhibited IL-2-reactive T cell generation, and the inhibition was sufficient to cause the reduction in T cell mitogenesis. A general relationship was observed between immunosuppressive activity of a given M phi and its active oxygen-producing ability (measured in terms of chemiluminescence) in response to phorbol myristate acetate triggering (r = .84, P less than .005). However, the suppressor activity of test M phi s was not reduced by superoxide dismutase and catalase, indicating that active oxygen radicals themselves did not mediate the expression of the immunosuppressive activity of these M phi s. On the other ahnd, indomethacin (an inhibitor of prostaglandin synthesis) caused a partial reduction in their immunosuppressive activity. The suppressor activity of M phi s induced with intraperitoneal injection of recombinant interferon gamma (IFN-gamma) was markedly reduced in the presence of myoglobin, a scavenger for nitric oxide radical (NO.). Tumor necrosis factor alpha (TNF alpha) failed to affect Con A mitogenesis of splenic T cells, even in combination with IFN-gamma. On the other hand, unsaturated long-chain fatty acids including oleic, linoleic, linolenic, and arachidonic acids markedly reduced the T cell function. These findings suggest some important roles of prostaglandins, NO., and long-chain unsaturated fatty acids as mediators of the expression of immunosuppressive function of the peritoneal M phi s.