A slow reacting substance is produced when mixed peritoneal cells from rats are incubated with low concentrations of the calcium ionophore A 23187 in the presence of 0.01 M cysteine. The product resembles slow reacting substance of anaphylaxis (SRS-A), if it is not identical to it, based on its behavior during chromatography on Amberlite XAD-2, silicic acid, and Sephadex LH-20; its destruction by dilute HCl and its stability in dilute alkali; its susceptibility to inactivation by aryl sulfatase; and its smooth muscle contracting activity on the guinea pig ileum and the gerbil colon. In vivo selective depletion procedures and in vitro cell isolation procedures were employed to identify the cells responsible for SRS-A production. Mast cells and mixtures of neutrophils and eosinophils produced only very small amounts of SRS-A; this could not be explained by the destruction of preformed SRS-A by these cells. The low yield of SRS-A from mast cells was also not explained by feedback inhibition caused by the histamine they released, because high concentrations of the H2 antagonist, Burimamide, caused only modest increases in SRS-A production by mast cell-containing cell suspensions. Granulocyte-depleted cell suspensions which were derived from thioglycolate-induced mononuclear cell exudates produced more SRS-A per cell than did comparable preparations from uninduced rats. The SRS-A production by induced monocytes was virtually abolished in tandem with the depletion of the phagocytic cells in the preparation. The possible implications of these findings on the source and role of SRS-A in the rat are briefly considered.