Activins are members of the Transforming Growth Factor‐β superfamily. This superfamily of secreted growth factors activates transmembrane serine‐threonine kinase receptors, thereby stimulating Smad‐mediated intracellular signaling cascades. These cascades alter the gene expression profile of the target cells. Follistatin (FST) is a protein antagonist that directly binds to activins and other TGF‐β‐like proteins, preventing their interaction with cell‐surface receptors. Alternative splicing and proteolysis result in three isoforms of FST which differ in their C‐terminal regions. The three FST isoforms have different affinities for activins, as well as the extracellular matrix component heparan sulfate. We are focused on two FST isoforms, FST315 and FST288. FST315 contains an acidic C‐terminal region that is absent in FST288. Crystal structures of FST315 and FST288 with activin A are unable to explain the basis for isoform‐specific effects. The structures are similar and the acidic C‐terminal region of FST315 is not observed in the electron density. To identify differences between FST315 and FST288, we have used hydrogen/deuterium exchange coupled to mass spectrometry. We compare exchange of FST315 and FST288, both alone and in complex with activin A. The results will improve our molecular understanding of how FST isoforms regulate signaling by activin A.Support or Funding InformationStartup funds to S. D'Arcy from The University of Texas at Dallas.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.