Nonparenchymal liver cells from untreated C3HeB/FeJ mice, when incubated in medium containing-10% fetal bovine serum or portal serum, produced significant amounts of interferon alpha/beta (IFNc~/[3). In contrast, other cell populations (spleen, mononuclear blood cells and peritoneal cells) from C3HeB/FeJ mice or nonparenchymal liver cells from other strains of mice (C3H/ HeJ, germ-free C 3 H / H e N and C57B1/6J) produced little or no detectable IFN in fetal bovine serum under the same culture conditions. The cells in the nonparenchymal liver cell population responsible for IFNc~/[~ production were adherent, phagocytic, silica-sensitive, carbonyl-iron-sensitive, and Thyl.2-, presumably Kupffer cells or resident liver macrophages. IFNcc~3 production by cultured Kupffer cells was not observed if medium containing fetal bovine serum or portal serum was treated with polymyxin B or if Kupffer cells were cultured in serum-free medium. This suggested that small amounts of endotoxin in fetal bovine or portal serum stimulated Kupffer cells to produce IFNc~/13. Possibly, Kupffer ceils are in a different state of activation/maturation than peritoneal and splenic macrophages since the sensitivity of resident Kupffer cells from C3HeB/FeJ mice to the stimulatory effects of endotoxin. The endogenous production of IFNc~/[3 by Kupffer cells from C3HeB/FeJ mice can augment liver-associated natural killer (NK) activity against YAC-1 cells (4 h) and induce liver-associated cytotoxic activity, not restricted by the major histocompatibility complex, against NK resistant P815 mastocytoma cells (18 h).