The metabolic fate of [U-14C]glucose has been examined in detail in adult rat hepatocytes in primary monolayer culture, as well as in two permanent cell lines--Buffalo rat liver (BRL) and transplantable rat hepatoma (HTC) cells-derived from normal rat liver and from rat hepatoma, respectively. Under defined conditions of incubation, at a glucose concentration of 5.5 mM, the three types of cultured liver cells exhibited pronounced differences in glucose metabolism. Primary cultures, like the intact liver, differed from the cell lines in consuming relatively small amounts of glucose and converting approximately 50% of the total metabolized glucose to lactate. By contrast, the permantent cell lines consumed glucose at a 40-fold greater rate than did primary cultures, converting 80--90% of the carbohydrate to lactate. Oxidative metabolism of glucose carbon also differed among the three types of liver culture. Of the total [U-14C]glucose consumed, primary cultures converted approximately 30% to labeled CO2 per hour, whereas the liver cell lines converted 5--10%. Finally, glucose metabolism in primary culture exhibited adaptation as hepatocytes aged in culture, shifting progressively toward the pattern exhibited by the permanent cell lines. This change occurred over a time course similar to that for other kinds of functional change in hepatocytes in primary culture and thus may be relevant to the general problem of phenotypic alteration in liver cell culture.