To elucidate the regulation of follistatin production in the liver, we studied changes in steady-state follistatin mRNA levels in cultured rat hepatocytes. Activin A stimulated follistatin mRNA levels in a time- and concentration-dependent manner. The stimulatory effect of activin A on follistatin mRNA was significant at 2 h, maximal at 6 h and declined thereafter. Incubating the cells with EGF increased follistatin mRNA levels at 48 h and later. The EGF-induced increase in follistatin mRNA was markedly inhibited by exogenous follistatin in the culture medium, which blocks the action of activin A synthesized in hepatocytes, suggesting that endogenous activin A at least partly mediated the effect of EGF. We also examined the effects of transforming growth factor-beta (TGF-beta), glucagon and alpha-adrenergic agonist, phenylephrine, on follistatin mRNA levels. TGF-beta increased the follistatin mRNA to levels similar to those caused by activin A. Phenylephrine and glucagon also increased follistatin mRNA levels but the effects were transient and weaker than those caused by activin A. Finally, follistatin mRNA levels were markedly increased in remnant liver 3 h after 70% hepatectomy. The mRNA remained elevated for up to 72 h. These results indicate that the expression of mRNA for follistatin is positively controlled by activin A, TGF-beta and other hormones or neurotransmitters. The stimulatory effect of EGF on follistatin mRNA is mediated by activin A released from hepatocytes.