We hypothesized that superoxide from Kupffer cells (KC) contributes to hepatocarcinogenesis. p47phox^−/− mice, deficient in phagocyte NADPH oxidase and superoxide generation, received a single dose of the hepatocarcinogen diethylnitrosamine (DEN). The following hepatic effects were observed at time points between 30 min and 35 days. Liver damage after DEN was manifested by loss of body and liver mass and of liver DNA and by an increase in apoptosis, necrosis and signs of inflammation. These effects were massive in wild-type ( wt ) male mice, but only very mild in p47phox^−/− mice. Regenerative DNA synthesis subsequent to liver damage was high in wt male mice, but weak in p47phox^−/− mice. In females the apparent protection by p47phox^−/− was less pronounced than in males. Therefore, further experiments were performed with males. In KC isolated from wt mice superoxide production was enhanced by DEN pretreatment in vivo . Also, in vitro addition of DEN to KC cultures induced superoxide release, similarly to lipopolysaccharide, a standard KC activator. Thus, DEN directly activates wt KC to produce superoxide. KC from p47phox^−/− mice did not release superoxide. TNFα production by isolated KC was transiently depressed 0.5 h after DEN treatment in vivo , but recovered rapidly. In blood serum TNFα levels of wt mice were elevated for the initial 6 h. TNFα in KC cultures and in serum of p47phox^−/− mice was reduced. DEN in vivo induced DNA damage (‘comets’) in hepatocytes. This damage was extensive in wt mice but much less in p47phox^−/− mice. These studies suggest two conclusions: (i) superoxide generation by phagocytes during liver damage and inflammation aggravates genotoxic and cytotoxic effects in hepatocytes and may thus contribute to tumor initiation and promotion; (ii) DEN has a direct stimulatory effect on KC to release superoxide and TNFα.