Follistatin binds and neutralizes members of the TGFbeta superfamily including activin, myostatin, and growth and differentiation factor 11 (GDF11). Crystal structure analysis of the follistatin-activin complex revealed extensive contacts between follistatin domain (FSD)-2 and activin that was critical for the high-affinity interaction. However, it remained unknown whether follistatin residues involved with myostatin and GDF11 binding were distinct from those involved with activin binding. If so, this would allow development of myostatin antagonists that would not inhibit activin actions, a desirable feature for development of myostatin antagonists for treatment of muscle-wasting disorders. We tested this hypothesis with our panel of point and domain swapping follistatin mutants using competitive binding analyses and in vitro bioassays. Our results demonstrate that activin binding and neutralization are mediated primarily by FSD2, whereas myostatin binding is more dependent on FSD1, such that deletion of FSD2 or adding an extra FSD1 in place of FSD2 creates myostatin antagonists with vastly reduced activin antagonism. However, these mutants also bind GDF11, indicating that further analysis is required for creation of myostatin antagonists that will not affect GDF11 activity that could potentially elicit GDF11-induced side effects in vivo.