The mononuclear cells in peritoneal washings from normal rats can be induced to produce large amounts of slow reacting substance of anaphylaxis by incubation with 10 mM cysteine in the presence of the calcium ionophore A-23187. This production of slow reacting substance could be inhibited by the addition of non-steroidal anti-inflammatory drugs, e.g., indomethacin, ibuprofen and flurbiprofen, Furthermore, mediator production was inhibited by eicosatetraynoic acid, the substrate analog of arachidonic acid, and by 9,11-azoprosta-5, 13-dienoic acid (AZO analog 1), a structural analog of the prostaglandin endoperoxide, PGH2, which known to inhibit thromboxane synthesis. Relatively high concentrations of hydrocortisone acetate inhibited mediator production; this inhibition could be partly reversed by the addition of arachidonic acid or to a lesser extent by eicosatrienoic acid. Preliminary results suggest that a small fraction of the 3H-labled arachidonic acid which was taken up by these cells in vitro was associated with slow reacting substance. We postulate that slow reacting substance of anaphylaxis may be derived from a prostaglandin endoperoxide which is formed during the oxidation of arachidonic acid by the prostaglandin fatty acid cyclooxygenase.