1. A new support system has been used which provides long-term maintenance of rat liver parenchymal cells in monolayer cultures. The cells, maintained on collagen gel/polychlorinated vinylidene film, expressed aminopyrine metabolizing activity for up to 5 days. This culture system was utilized to study the metabolism of aminopyrine in the liver cells isolated from normal, alloxan- and streptozotocin-diabetic rats. 2. Aminopyrine was metabolized at a slower rate in both types of cultured diabetic rat hepatocytes than in cultured normal rat hepatocytes, as judged from higher levels of the unchanged drug in the culture medium. 3. The formation of the metabolites 4-monoaminoantipyrine, 4-acetylaminoantipyrine and 4-formylaminoantipyrine decreased in the cultured diabetic rat hepatocytes, while that of 4-aminoantipyrine was at the same levels as controls. In contrast, 3-hydroxymethyl-2-methyl-4-dimethylamino-1-phenyl-3-pyrazolin-5-on e (AM-CH2OH) formation in the cultured diabetic rat hepatocytes increased over control value. These findings agree with in vivo results which have been reported by the authors. 4. The increase in AM-CH2OH was prevented by insulin in a dose-dependent manner. However, insulin did not affect the formation of other metabolites. These findings indicate that the amount of cytochrome P-450 isozyme involved in the oxidation of 3-methyl group may be regulated by insulin. 5. The present results, indicate that this primary culture system is a useful tool for the study of drug metabolism in diabetes.