Leukocytic pyrogen (LP) is a polypeptide product of activated mononuclear phagocytes that has been established as the endogenous mediator of fever. Recent studies show LP may be identical to lymphocyte-activating factor (LAF or interleukin 1). In the present experiments, chromatographic separations of human LP indicate fractions that produce fever in rabbits and enhance phytohemagglutinin- (PHA) induced murine thymocyte proliferation (LAF activity) also stimulate human monocyte monolayers to release prostaglandin E (PGE). Ibuprofen, an inhibitor of the cyclooxygenase pathway of arachidonic acid metabolism, prevents PGE release from monocytes stimulated with LP and also brings about a rapid defervesence of LP-induced fever in rabbits. Concentrations of ibuprofen that block PGE production in vitro and in vivo, however, have no effect on LP-induced thymocyte proliferation. When the arachidonic acid analogue eicosa-5,8,11,14-tetraynoic acid was added to thymocytes in the LAF assay, a 50 to 70% decrease in LP-induced enhancement of proliferation was observed without a decrease in the background PHA response. Similar results were also observed with BW755C, a water-soluble inhibitor of the lipoxygenase pathway. The results of these studies suggest products of the cyclooxygenase pathway of arachidonate metabolism are not involved in the mechanism by which LP stimulates thymocyte proliferation. On the other hand, the results suggest products of the lipoxygenase pathway may mediate the thymocyte proliferative response induced by LP.