In this study the authors analyzed ethanol (Et-OH) and acetaldehyde (Ac-CHO) metabolism in cultured hepatocytes isolated from chronic alcoholic rats. Hepatocytes were isolated and cultured from two groups of rats, one was fed with a liquid diet containing Et-OH and another was pair-fed with a control diet for 4 weeks. After 48 hours of the primary culture, Et-OH was added to the culture medium at a final concentration of 5 mM with or without 2 mM 4-methyl pyrazole (Py). Serial changes of Et-OH and Ac-CHO levels in the medium for 48 hours were determined in 4 groups of the alcohol alone, alcohol-Py, control and control-Py groups. In the alcohol alone and control groups, Et-OH disappearance rates (EDR), which are roughly equivalent to Et-OH oxidation rates in the hepatocytes, were significantly higher than those in the corresponding Py treated groups. In the two alcoholic groups, the EDR was significantly higher than those in the corresponding control groups. In cultured hepatocytes, 75-80% of Ac-CHO produced from Et-OH was oxidized. The increasing rates of Ac-CHO (AcICR), a function of Ac-CHO production and oxidation rates in the hepatocytes, increased in parallel with the increase in the EDR. However, the AcICR/EDR rate, which is a parameter of the Ac-CHO oxidation rate in the hepatocytes, was not different among the 4 groups. These results indicate that the cultured cells maintain the characteristics of Et-OH metabolism in chronic alcoholic rats and may also be used for the study of Et-OH and Ac-CHO metabolism as an in vitro model.