Hepatocytes from adult rats, cultivated for 4 days in RPMI 1640 medium, were used for studies on mechanisms of induction of ornithine decarboxylase (ODC). The activity of ODC was measured partially in situ by an assay method using (3)H-labelled ornithine and analysing of the labelled putrescine formed. The tumour-promoting phorbol ester 12-O-tetradecanoyl-13-acetate (TPA) induced ODC by several hundred percent in this system. No ODC activity remained after the cells were incubated for 4 hr with both TPA and cycloheximide, and no induction of ODC occurred when actinomycin D and TPA were present, although some ODC activity remained. Experiments with cyclohexidine and Actinomycin D (Act. D), respectively, indicated that the induction of ODC by TPA was totally dependent on protein synthesis and was also dependent on transcriptional events. Sphingosine and H-7 (1-(5-isoquinolinyl-sulphonyl)-2-methylpiperazine) inhibited the induction of ODC by TPA indicating the involvement of protein kinase C (PK-C) in this process. Pre-incubation of the hepatocytes with TPA, a procedure which down-regulates PK-C, decreased the induction of ODC by a subsequent new exposure to TPA, but had no effect on the induction of ODC caused by asparagine in this system. The induction caused by asparagine was additive to that caused by TPA. Studies with copper (diisopropylsalicylate)(2) and manganese(IV) desferrioxamine, and with prostaglandin D(2), respectively, gave no indication that activated oxygen or prostaglandins were involved in the induction of ODC by TPA. The results of this study are generally consistent with previously published in vivo data and show that a well defined hepatocyte in vitro system is suitable for mechanistic studies of ODC induction.