Activins are formed by dimerization of β-subunits and, as members of the TGF-β superfamily, have diverse roles as potent growth and differentiation factors. As the biological function of the activin C homodimer (βC-βC) is unknown, we sought to compare activin A (βA-βA), B (βB-β B), and C homodimer bioactivities and to investigate the consequences of activin βC-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 βC-subunit heterodimerized with activin βA in vitro to form activin AC. Therefore, we hypothesize that the activin β 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 ELISA that we developed for this purpose. In the PC3 human prostate tumor cell line, activin βC-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 β C-subunit as a regulatory mechanism to reduce activin A secretion via intracellular heterodimerization.