Tumor necrosis factor (TNF) has been implicated as one of the mediators of the immunologic and metabolic changes in endotoxemia. Under adverse conditions, TNF can also be cytotoxic, and its effects can ultimately contribute to organ failure. This study shows that a 30-min infusion of a nonlethal dose of TNF induced the release of superoxide anion (0.9 nmol.min-1.g-1) by the in situ perfused rat liver. TNF also primed the liver to generate more superoxide anion (2.0 nmol.min-1.g-1) in response to an in vitro challenge with phorbol 12-myristate 13-acetate (PMA). Kupffer cells are most likely responsible for the superoxide anion production under these conditions, because the isolated Kupffer cells from TNF-infused rats produced increased quantities of superoxide anion (4-8 nmol/10(6) cells) when subsequently treated in vitro with either PMA or opsonized zymosan (control less than 1 nmol/10(6) cells). Thus, under these experimental conditions, TNF in vivo primed the Kupffer cells, but not the hepatocytes, endothelial cells, and the blood or hepatic neutrophils, to release more superoxide anion. These studies indicate that during a short-term nonlethal TNF infusion, Kupffer cells are a major target of TNF action, leading to the release of toxic-oxygen metabolites that may contribute to organ failure.