The present study was conducted to determine the role of two autocrine factors, activin A and transforming growth factor β (TGF-β), in the growth regulation of AML12 hepatocytes. We overexpressed truncated type II activin and/or TGF-β receptors in AML12 cells. In AML12 cells overexpressing truncated type II activin receptors (AML-tAR cells), the inhibitory effect of activin A on DNA synthesis was completely blocked. AML-tAR cells proliferated faster than parental cells, both in the presence and absence of epidermal growth factor (EGF). However, AML-tAR cells could not grow in soft agar. Follistatin augmented EGF-induced DNA synthesis in AML12 cells, whereas it was ineffective in AML-tAR cells. In AML12 cells overexpressing truncated type II TGF-β receptor (AML-tTR cells), the inhibitory effect of TGF-β on DNA synthesis was blocked. AML-tTR cells proliferated faster than parental cells, both in the presence and absence of EGF, but at a slower rate than that of AML-tAR cells. AML-tTR cells did not grow in soft agar. The growth rate of cells overexpressing both types of truncated receptors was identical to that of AML-tAR cells, and these cells did not grow in soft agar. These results indicate that both activin A and TGF-β act as autocrine inhibitors of DNA synthesis in AML12 cells, and that the blocking of the actions of two factors does not lead to transformation. Activin A is a predominant autocrine factor in these cells.