The effect of activins AB and B on DNA synthesis stimulated by epidermal growth factor (EGF) was studied in primary cultured rat hepatocytes and compared with the effect of activin A, a suppressor of DNA synthesis. Activin AB inhibited DNA synthesis as assessed by [3H]thymidine incorporation. The inhibition by activin AB was detected at 6 ng/ml, and the 12.5 ng/ml concentration produced almost maximal inhibition, approximately 40%, almost the same as that produced by activin A. Inhibition by activin A was detected at 3 ng/ml, and the 6 ng/ml concentration produced almost maximal inhibition. Activin B, on the other hand, had no effect on DNA synthesis up to 50 ng/ml. The increase in labeling index by EGF was also reduced to about 20% by 25 ng/ml activin A and activin AB, but not by activin B. Activin B, however, inhibited the binding of [125I]activin A to hepatocytes, but had no effect on the inhibition of DNA synthesis by activin A, even at 3-fold excess concentrations. These findings suggest that activin AB may act in the same manner as activin A does in terms EGF's inhibitory effect on DNA synthesis, although the effective concentration is higher than that of activin A. The findings also suggest that activin B receptors are present in hepatocytes but that they do not mediate signal transduction leading to the inhibition of DNA synthesis.