Activins are formed by dimerization of subunits and, as members of the TGF- superfamily, have diverse roles as potent growth and differentiation factors. Since the biological function of the activin C homodimer (_C-_C) is unknown, we sought to compare activin A (_A-_A), B(_B-_B) and C homodimer bioactivity and to investigate the consequences of activin _C subunit overexpression in prostate tumor cells. Exogenous activin A and B homodimers inhibited cell growth and activated activin-responsive promoters. In contrast, the activin C homodimer was unable to elicit these responses. We previously showed that the activin _C subunit heterodimerised with activin _A in vitro to form activin AC. Therefore, we hypothesize that the activin _C subunit regulates the levels of bioactive activin A by the formation of activin AC heterodimers. To test this hypothesis we measured activin AC heterodimer production using a novel specific two-site enzyme linked immunosorbent assay (ELISA) that we developed for this purpose. In the PC3 human prostate tumor cell line, activin _C subunit overexpression increased activin AC heterodimer levels, concomitantly reduced activin A levels and decreased activin signaling. Overall, these data are consistent with a role for the activin _C subunit as a regulatory mechanism to reduce activin A secretion via intracellular heterodimerization.