The biological chemistry of nitric oxide (NO) in the oxygenated cellular environment is extremely complex. It involves the direct interaction of NO with specific biomolecules and the so-called indirect effects, due to secondary more potent oxidant species derived from NO which are also able to react with DNA, lipids, thiols and transition metals (Wink et al., 1996; Nathan, 1992). In addition to its regulatory role as a signalling molecule (Nathan, 1992; Moncada and Palmer, 1991) it has become evident that NO (or NO-derived species) is a critical factor involved in various toxicological mechanisms (Wink et al., 1996; Wang et al., 1998; Estevez et al., 1999; Wink et al., 1999). Some controversy exists however about the damaging vs. protective actions of NO on oxidative injury, whose biological significance in living cells and tissues remains still ill defined. Research in this laboratory (López-García, 1998; López-García and Sanz-Gonzalez, 2000) has shown that NO synthesis is significantly activated in hepatocytes from control rats following isolation by the classical collagenase-based procedure. NO overproduction appears to be due to the very early activation of liver constitutive Ca2+-dependent NO synthase (cNOS). Previous results have also provided first experimental evidence for the direct involvement of endogenously generated NO as a causal factor responsible for important phenotypic changes commonly observed in short-term cultured hepatocytes, which includes the early impairment of hepatocyte mitochondrial function--i.e., transient cell energy depletion--and glucose metabolism, and the well-known quick and irreversible loss of P450 content (López et al. 1987; López-García, 1998). This study aims to further characterise the mechanisms underlying this phenomenon. Results show that the hepatocyte isolation procedure (the commonly employed collagenase-based two step liver perfusion method) induces strong oxidative stress that lasts for at least 4 h in culture and involves both oxygen-derived (ROS) and nitrogen-derived (RNS) reactive species. On the basis of the combined use of dihydrorhodamine 123 (DHR) as a probe and L-NAME (N(G)-nitro-L-arginine methyl ester) to efficiently block NO synthesis, the analysis of the amount, the time-course pattern, and the nature of the species involved support the view that peroxynitrite* (PN) is readily formed within the early culture hours. Immunodetection of protein bound 3-nitrotyrosine provides direct evidence for PN generation upon hepatocyte isolation: several nitrated protein bands--most already present after only 30 min of liver perfusion and quantitatively increasing for the first 2 hours in culture--have been identified as preferential PN protein targets in the different cellular compartments. Since the early inhibition of NO synthesis is enough to provide full maintenance of the hepatocyte initial P450 content, results support the view that PN--while not affecting cell viability and monolayer development--is the main species likely responsible for the early loss of P450 in short-term cultured hepatocytes.