Peroxisome proliferators (PPs) cause hepatocyte proliferation and tumorigenesis in rodent liver. PPs induce hepatocyte DNA synthesis although the mechanism is unclear. Tumour necrosis factor (alpha) (TNF(alpha)) and epidermal growth factor (EGF) have been implicated in mediating this growth response since these factors induce a threefold and 17.2-fold increase, respectively, in DNA synthesis in rat primary hepatocyte cultures. Previously, others have suggested that TNF(alpha) acts as a primer to sensitise hepatocytes to the proliferative effects of growth factors. Indeed, here we show that costimulation with TNF(alpha) and a suboptimal (4-20% of optimal) concentration of EGF permits an 11.7-fold increase in DNA synthesis in rat primary hepatocyte cultures. The PP nafenopin induced a 2. 3-fold increase in DNA synthesis but there was no further increase upon co-administration of either TNF(alpha) or a suboptimal concentration of EGF. Furthermore, there was no gross dysregulation of the CDK and cyclin protein expression profile upon stimulation with nafenopin. Using a specific epidermal growth factor receptor tyrosine kinase inhibitor (4-(3-chloro-4-fluorophenylamino)-7-methoxy-6- (3-¿1-pyrolidino)-propoxyquinazoline, EGFR-TKI), we show that signalling through EGF-R is not required for nafenopin-induced DNA synthesis. The EGFR-TKI also prevented progression into S phase upon stimulation with TNF(alpha), but DNA synthesis was not reduced to control levels, indicating that TNF(alpha) has a mitogenic activity in the absence of EGF signalling. Therefore, although TNF(alpha) can act as a priming factor for growth factors such as EGF, nafenopin does not appear to act via this mechanism.