We reported a rat model of necrotizing enterocolitis by injecting platelet-activating factor (PAF) into the mesenteric vascular bed, and suggested that leukotrienes (LT) are secondary mediators. The present study, using isolated, buffer-perfused rat small intestine, shows: Isolated, perfused small intestine synthesizes LTs in response to PAF. Leukotriene C4 (LTC4) was the predominant LT released. The initial vasoconstriction after PAF injection was due to a transient release of LTC4 since FPL 55712 pretreatment abolished the vasoconstriction. The sustained rise in perfusion pressure was also blocked by FPL 55712, which suggests that other vasoconstrictors released are regulated by LTs. The vasoconstrictor(s) responsible for sustained rise in perfusion pressure is unknown, but is not thromboxane. Most of the LT was released from intestinal tissue rather than mesenteric arteries. Vasodilating prostaglandins (PGs) were also released, probably secondary to LTs. The complex interaction of these lipid mediators (PAF, LTs, and PGs) and their subtle balance may affect the course of the disease.