Prostaglandin E2 (PGE2) modulates a variety of physiological processes including the production of inflammatory cytokines. There are two cyclooxygenase (Cox) enzymes, Cox-1 and Cox-2, that are responsible for initiating PGE2 synthesis. These isozymes catalyze identical biosynthetic reactions but are regulated by different mechanisms in the cell. This report examines differences in the roles of Cox-1 and Cox-2 in regulating cytokine synthesis in macrophages. We employed agents that selectively modulate the activity of each isozyme and measured their effects on synthesis of interleukin (IL)-6, IL-1, and tumor necrosis factor-alpha by peritoneal macrophages. Among these three cytokines, only IL-6 synthesis was stimulated by production of endogenous PGE2. This effect was specifically linked to activation of Cox-2 and not Cox-1. The specificity derives, partly, from the timing of the production of PGE2 following stimulation of each isozyme and from induction of ancillary signals that control the response to PGE2. The experimental findings demonstrate that the effects of Cox-1 and Cox-2 activity on macrophage IL-6 synthesis are segregated. This provides a mechanism for IL-6 to be induced selectively during inflammation.