The metabolism of 1,3-cyclohexadiene by hepatocytes from phenobarbital induced rat has been investigated. Parenchymal cells were obtained by liver perfusion with a hyaluronidase-collagenase mixture. The addition of the diene to a suspension of hepatocytes gave rise to a type I difference spectrum indicating the formation of an enzyme-substrate complex with cytochrome P-450. The subsequent metabolic pathway of 1,3-cyclohexadiene has been shown to involve, as the first step, the formation of 1,2-epoxy-3-cyclohexene, which is rapidly hydrolyzed to trans-3-cyclohexene-1,2-diol and trans-2-cyclohexene-1,4-diol by a non-enzymatic process. The monoepoxide could not be detected in the incubation medium because of its high reactivity. Therefore, kinetic parameters of the epoxidation reaction were determined by following the rate of production of the diols. When incubated with hepatocytes, trans-3-cyclohexene-1,2-diol, the main product of 1,3-cyclohexadiene metabolism, elicited a reverse type I spectrum, indicating that this compound is not a good substrate for the monooxygenase system.