Mouse resident peritoneal macrophages incubated with ionophore A23187 or opsonized zymosan released leukotrienes (LT) B4 and C4 (LTB4 and LTC4) and LTC4 and LTD4, respectively. In contrast, incubation with Toxoplasma gondii, an obligate intracellular protozoan, led to the formation of 11-, 12-, and 15-hydroxyicosatetraenoic acids (HETEs), together with an unidentified compound, designated compound X. Each of these compounds incorporated [3H]arachidonic acid from the macrophage during phagocytosis of T. gondii. Compound X migrated immediately prior to 15-HETE by reverse-phase HPLC and was distinct from authentic monoHETE, monohydroperoxyicosatetraenoic acid (mono-HPETE), and dihydroxyicosatetraenoic acid (diHETE) standards. The generation of compound X by macrophages correlated with the extent of phagocytosis of T. gondii and with intracellular survival of the organisms. Prior antibody-coating of T. gondii or activation of macrophages, either of which inhibited survival and replication of ingested organisms, was associated with production of LTD4 but not compound X. Killed organisms also stimulated LTD4 release only. Although T. gondii concentrated arachidonic acid, they did not metabolize the compound to identifiable lipoxygenase products. Preincubation of macrophages with the relative lipoxygenase inhibitors nordihydroguaiaretic acid or 5,8,11,14-icosatetraynoic acid inhibited the formation of compound X. The absence of leukotriene production by macrophages ingesting T. gondii may explain the relative lack of a neutrophil inflammatory response in diseases due to obligate intracellular organisms. Alternatively, compound X may have functional activities that might mediate some of the host responses to cellular parasitism.