Kupffer cells are the largest population of fixed tissue macrophages in the body and produce a number of mediators that are involved in host defense. These mediators include cytokines such as tumor necrosis factor-δ and interleukin-1, prostaglandins, oxygen radicals, and nitric oxide. Prostaglandins are produced by adjacent endothelial cells in addition to Kupffer cells and regulate a number of cellular functions in a wide array of cells, but their role in nitric oxide synthesis is controversial. We studied the role of prostaglandins in regulating lipopolysaccharide (LPS)-induced nitric oxide synthesis in cultured rat Kupffer cells. Prostaglandin E2 (PGE2) inhibited Kupffer cell nitric oxide synthesis in a dose-dependent fashion in both 24- and 48-hr cultures. The effect of PGE2 persisted at low and high LPS concentrations. Prostaglandin analogues as well as other prostanoids also inhibited Kupffer cell nitric oxide synthesis. These data show that exogenous prostaglandins suppress Kupffer cell nitric oxide synthesis and may represent an important endogenous regulator of nitric oxide production.