Tolbutamide (TB; 1-butyl-3-p-tolylsulfonylurea) was used for metabolism studies with hepatocytes isolated from the rat, rabbit, dog, and squirrel monkey to validate their usefulness as models for comparative in vivo metabolism. Hepatocytes were prepared by whole liver or biopsy perfusion. TB (3 X 10(-4) M) was metabolized by each of the preparations over a 4-hr incubation period at rates ranging from 175.8 to 9.2 pmol/10(6) cells/min with the rates from hepatocytes from: rat greater than rabbit much greater than squirrel monkey greater than dog. The metabolite profiles determined in extracts of hepatocyte suspensions after 4 hr of incubation showed marked species differences. The major metabolite for the rat and squirrel monkey was 1-butyl-3-p-hydroxy-methylphenylsulfonylurea (73.2 and 46.7% of total metabolites, respectively). p-Tolylsulfonylurea and p-tolylsulfonamide were the major metabolites found in the dog (44.1 and 40.2%, respectively). Rabbit hepatocytes formed mostly 1-butyl-3-p-carboxyphenylsulfonylurea (63.9%). Both the relative rates of metabolism and the metabolite profiles from hepatocytes from the rat, rabbit, and dog correlated well with published in vivo data on TB plasma half-lives and urinary metabolite profiles. These results suggest that isolated hepatocytes may have utility as in vitro models for comparative in vivo metabolism.