Well-differentiated Reuber H35 rat hepatoma cells in culture maintain a variety of biochemical functions characteristic of hepatocytes [Deschatrette, J., and M. C. Weiss. 1974. Biochimie. 56: 1603-1611]. To demonstrate the suitability of this system as a model for exploring mechanisms of ethanol hepatotoxicity, the following were investigated: 1) ethanol metabolism in whole cells and cell extracts and 2) effects of ethanol exposure on cellular lipid content. Cultures of H35 cells exposed to 10 mm ethanol metabolized the ethanol at rates similar to those reported in rat liver. Under these conditions, soluble alcohol dehydrogenase activity accounted for greater than 87% of total ethanol metabolism. H35 cells exposed to 240 mm ethanol for 3 days contained four times more triacylglycerol and cholesteryl ester than control cells. Total phospholipid and unesterified cholesterol levels were unaffected by ethanol. Neutral lipid content of Chinese hamster ovary cells was unchanged after ethanol exposure. The increased triacylglycerol content of ethanol-treated H35 cells appeared to result from an accelerated rate of conversion of long chain fatty acids into triacylglycerol. Several lines of evidence indicated that alcohol dehydrogenase-mediated ethanol oxidation was critical in promoting increased triacylglycerol content of cultured cells. Since 240 mm ethanol blocked cellular proliferation, long term effects of ethanol were studied at a level of 10 mm, which allowed a nearly normal growth rate. After 7 weeks of continuous exposure, 10 mm ethanol-treated H35 cells contained five times more triacylglycerol than paired controls. The well-differentiated H35 cell appears to be an excellent in vitro model system for studying both short-term and long-term effects of ethanol on liver cells.-Polokoff, M. A., M. Iwahashi, and F. R. Simon. Ethanol treatment increases triacylglycerol and cholesteryl ester content of cultured hepatoma cells.