1. A method for metabolic studies with isolated rat hepatocytes has been developed. The rate of metabolism of a number of barbiturates has been studied both in hepatocytes and in 9000 g rat liver supernatant suspension. 2. The half-lives and clearance values, relative to heptabarbital, determined in hepatocytes were similar to the values obtained with isolated perfused rat liver. The values obtained with the 9000 g supernatant preparation were not. 3. In respect of metabolites formed, the metabolism of barbiturates in hepatocytes is comparable to that of the intact rat or liver perfusion. The metabolism in the 9000 g supernatant, however, is not.