Carnitine and acetylcarnitine are used as dietary supplements and as therapeutic agents. Carnitine attenuates ethanol metabolism in intact animals but the in vitro activities of alcohol dehydrogenase (ADH), microsomal ethanol oxidizing system (MEOS) or catalase are not significantly altered by carnitine. Since acetylcarnitine was a far more potent inhibitor of ethanol oxidation than carnitine in hepatocytes, the activities of rat liver ADH and MEOS were determined with or without acetylcarnitine. The activity of ADH, not MEOS, was significantly inhibited by acetylcarnitine at NAD: acetylcarnitine < or = 1. The inhibition is of a competitive nature where acetylcarnitine competes with NAD+ (Ki = 135 mumol.L-1). This finding is unique in that this is the first report of this function of acetylcarnitine and it is a novel interaction between two important nutrients, niacin and carnitine.