The CYP3A subfamily is the most abundant of the human hepatic cytochrome P450 enzymes. They mediate the biotransformation of many drugs, including a number of psychotropic, cardiac, analgesic, hormonal, immunosuppressant, antineoplastic, and antihistaminic agents. We studied diet/ethanol interactions using total enteral nutrition in adult male Sprague-Dawley rats with diets containing 16% protein, ethanol (13 g/kg), corn oil (fat; 25-45%), and carbohydrate (CHO; 1-21%). Using this model, chronic ethanol feeding decreased CYP3A activity (testosterone 6 beta-hydroxylation) and apoprotein levels (Western blot) (P <.05) and these effects were independent of the dietary CHO/fat ratio. The CYP3A2 mRNA levels decreased (P <.05) in the rats fed ethanol-containing diets by 73 to 83% compared with rats fed control diets, regardless of the CHO/fat ratio. In contrast, ethanol induced CYP3A9 mRNA levels (P <.05) and this effect was greater (P <.05) in the high-CHO/low-fat group (11.3-fold) than in the low-CHO/high-fat group (2.6-fold). Purified recombinant rat P450 3A9 had a chlorzoxazone 6-hydroxylase activity with a turnover number 1.3 nmol/min/nmol of P450. These results indicate that 1) ethanol differentially affects the expression of CYP3A gene family and this regulation appears to be modulated by dietary CHO/fat ratio; 2) the decrease in testosterone 6 beta-hydroxylase activity and CYP3A apoprotein levels are most likely due to the ethanol-induced decrease in CYP3A2 mRNA levels; and 3) CYP3A9 is induced by ethanol and is a low-affinity, high-K(m) chlorzoxazone hydroxylase.