Macrophages have been described to release nitric oxide (NO) as a cytotoxic radical. This highly unstable substance is as well known as endothelium-derived relaxing factor produced by vascular endothelial cells. Because of its cytotoxic activity the synthesis of NO by rat Kupffer cells, the liver macrophages, upon stimulation with endotoxin (lipopolysaccharide; LPS) and tumor necrosis factor-alpha (TNF-alpha) in combination with prostaglandin E2 (PGE2) and dibutyryl cAMP (dBcAMP) was studied. Kupffer cells were stimulated after 48 h of primary culture. NO was quantified as NO2- in the cell medium 24 h after stimulation. LPS stimulated NO generation 5- to 10-fold over the basal level. This increase could be further enhanced by PGE2 and dBcAMP, especially when added 1 h after LPS. NO generation after stimulation with LPS or LPS + PGE2 depended on the simultaneous production of PGE2 by the stimulated Kupffer cells. It could be partly inhibited by anti-PGE2 antibody or acetylsalicylic acid. While murine TNF-alpha did not stimulate NO synthesis significantly, added PGE2 raised NO synthesis about 6-fold. The addition of dBcAMP to TNF-alpha in the same concentration as with LPS, however, had no effect. Thus, stimulation by LPS + PGE2 equals that of LPS + dBcAMP whereas TNF-alpha + PGE2 does not equal TNF-alpha + dBcAMP, indicating differences in the mode of action of PGE2 on LPS- or TNF-alpha-treated Kupffer cells.