Freshly isolated suspensions of rat parenchymal liver cells (hepatocytes) produce large amounts of nitrite following isolation. Nitrite production was inhibited by the inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine, as well as the transcription inhibitor actinomycin D. Increases in iNOS mRNA, protein, and activity levels correlated with the formation of nitrite. iNOS mRNA was first detectable 2 h after the onset of hepatocyte incubations and peaked at 4 h. These results indicate that nitrite formation is a result of the large scale production of nitric oxide (NO) by hepatocytes in response to the time-dependent induction of iNOS. NO production by hepatocytes was attenuated by pretreatment of rats with the Kupffer cell inhibitor, gadolinium chloride. Also, the addition of the endotoxin neutralizing agent, polymyxin B; the protein kinase inhibitor, staurosporine, and antioxidants to perfusion buffers and hepatocyte suspensions also decreased nitrite formation. Collectively, our results suggest that iNOS is induced in hepatocytes in response to the stresses generated during collagenase isolation procedures. The response appears to be triggered by a complex interaction between several different factors including Kupffer cell activation, reactive oxygen species generation, and endotoxin contamination of collagenase preparations.