Several agonists acting on G protein-coupled receptors (GPCR) enhance the mitogenic effect of epidermal growth factor (EGF) in rat hepatocytes, through mechanisms that have only partially been clarified. Results in various cells have led to the idea that a major mechanism for GPCR-mediated stimulation of cell growth is transactivation of receptor tyrosine kinases, particularly the EGF receptor (EGFR), leading to rapid phosphorylation of the EGFR and activation of downstream signaling pathways. In the present study cultured rat hepatocytes were exposed to various GPCR agonists, including vasopressin, angiotensin II (Ang.II), norepinephrine, or prostaglandin F(2 alpha) (PGF(2 alpha)). None of these agents increased the phosphorylation of the EGFR or the docking protein Shc. Furthermore, we examined the effect of the GPCR agonists on the expression of two early response genes believed to be involved in growth activation. The GPCR agonists increased the mRNA expression of c-myc, and also of activating transcription factor 3 (ATF3)/liver regeneration factor-1 (LRF-1), which is a novel finding. Finally, the selective EGFR inhibitor AG1478 did not suppress the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) or the induction of c-myc or ATF3/LRF-1 by the GPCR agonists, and did not prevent the comitogenic effects induced by these agents, while it blocked the effect of EGF on these responses. The results suggest that GPCR agonists induce expression of ATF3/LRF-1 and c-myc and exert comitogenic effects through mechanisms that do not require EGFR transactivation.