Isolated hepatocyte suspensions prepared by collagenase perfusion released high levels of nitrite into the extracellular medium during an 8-hr incubation. The release was time dependent, with increases first occurring by 4 hr and continuing throughout the remainder of the incubation period. Nitrite production was inhibited by the nitric oxide synthase (NOS) inhibitors aminoguanidine and N(G)-nitro-L-arginine methyl ester (L-NAME), indicating that the nitrite is derived from nitric oxide (NO) production from NOS activity. Nitrite production was not related to bacterial or Kupffer cell contamination. The protein synthesis inhibitor cycloheximide and the transcription inhibitor actinomycin D also prevented nitrite production by parenchymal hepatocytes. Calcium-independent NOS enzyme activity increased with incubation times, and this increase coincided with the observed increases in nitrite production. Our results suggest that NOS is induced following the isolation of hepatocytes, and this induction results in the formation of high levels of NO.