Cytochrome P-450 (CYP) isoenzymes have been incriminated in the toxicity and carcinogenicity of various xenobiotics in different tissues, but prior measurements of their activity in pancreatic microsomes have been disappointing. We now applied new isolation methods and a highly sensitive procedure to assay for the metabolism of p-nitrophenol and 7-ethoxyresorufin, specific substrates for CYP2E1 (2E1) and CYP1A1 (1A1), respectively. 2E1 and 1A1 content was estimated with high-resolution chemiluminescent Western blots using recombinant 2E1 and 1A1 as standards. We found that p-nitrophenol hydroxylase activity was 5.07 ± 0.66 and 1.50 ± 0.26 pmol/min/mg of protein in pancreatic microsomes of ethanol-fed and control rats, respectively. Chronic ethanol treatment increased 2E1 content in pancreatic microsomes 3.6-fold. Activity and content of 2E1 were also assessed in hepatic microsomes: specific activity (expressed per 2E1 content) was similar in pancreatic and hepatic microsomes. There was also an inductive effect of 3-methylcholanthrene (MC) on 1A1 in pancreatic microsomes. Pancreatic microsomal 7-ethoxyresorufin-O-dealkylation activity in MC-treated rats was 19.6 ± 1.7 pmol/min/mg of protein, 61-fold higher than in controls. MC treatment increased the 1A1 content in pancreatic microsomes 42-fold. These results demonstrate that, in pancreatic microsomes, ethanol and MC exert striking inductive effects on 2E1 and 1A1 activities, which could play a role in the pathogenesis of pancreatitis and/or pancreatic cancer.