The interaction of mast cells with other leukocytes during immediate hypersensitivity reactions was tested by in vivo and in vitro experiments. Intraperitoneal challenge of passively sensitized rats with antigen caused the production of peptidoleukotrienes, leukotriene (LT)B4, thromboxane (TX)B2, and 6-keto-prostaglandin (PG) F1 alpha in the peritoneal cavity. Pretreatment of the rats with thioglycollate i.p. markedly changed the amount of eicosanoids formed. When polymorphonuclear leukocytes were the predominant cell type in the peritoneal exudate, both LTC4 and 6-keto-PGF1 alpha were decreased by 75% each and TXB2 by 50%. When elicited macrophages were predominant, there was an additional reduction in LTC4 by 68% as compared with 18 hr after thioglycollate treatment, but no additional change in the other arachidonic acid metabolites. In vitro antigen challenge of passively sensitized mouse bone marrow-derived mast cells caused the release of LTC4, LTB4, 6-trans-LTB4, 5-hydroxyeicosatetraenoic (5-HETE), and TXB2. Exposure to antigen of these mast cells in the presence of resident peritoneal macrophages markedly altered eicosanoid formation. Early in the time course (2 to 15 min), macrophages markedly enhanced all 5-lipoxygenase products. However, later in the time course (30 to 120 min), these products were decreased. This decrease was reversed by catalase and superoxide dismutase, which suggests the involvement of oxygen radicals. These active oxygen species also seemed to be generated by mast cells, because these enzymes caused an increase in 5-lipoxygenase products when mast cells were challenged alone. RIA of cyclooxygenase products showed that mast cells released only TXB2 when stimulated with antigen. When they were stimulated in the presence of macrophages, TXB2 and also PGE2 and 6-keto-PGF1 alpha were synthesized. Therefore, macrophages probably contribute the PGE2 and 6-keto-PGF1 alpha. Because the same amount of TXB2 was generated whether macrophages were present or not, the mast cells seem to be the major source of this compound. These data indicate that macrophages and possibly polymorphonuclear leukocytes participate in immediate hypersensitivity reactions.