Hepatocytes of adult male rats were isolated by hepatic perfusion with a mixture of collagenase and hyaluronidase. This procedure gave a high yield of viable cells, as determined by trypan blue exclusion. The addition of 1,3-cyclohexadiene to a suspension of isolated liver cells gave rise to a type-I spectral change indicative of the formation of the cytochrome P-450-substrate complex. 1,3-cyclohexadiene was then incubated with hepatocytes for different times, to determine its biotransformation. Cyclohexadiene-1,2-diol resulted to be the main metabolite. It is proposed that the metabolic pathway of 1,3-cyclohexadiene involves the intermediate formation of an epoxide which is rapidly transformed to the correspondent diol.