Loss of P450 during the early hours of monolayer formation is known to be the more serious limitation of primary cultured hepatocytes as an adequate model for the study of drug metabolism, toxicity and P450 induction. This study reports that endogenous nitric oxide (NO) formation is activated shortly after isolation by the classical collagenase-based liver perfusion methods. Both rapid P450 loss and aerobic mitochondrial energy metabolism impairment -- with subsequent changes on glucose metabolism -- are directly related to the high local generation of the radical at this stage. These effects can be reverted by the sole addition of NO biosynthesis inhibitors during liver perfusion and early culture hours, which allows catalytically active P450 to be preserved at levels close to those of the intact liver.