Pyruvate formate-lyase-activating enzyme (PFL-AE) activates pyruvate formate-lyase (PFL) by generating a catalytically essential radical on Gly-734 of PFL. Crystal structures of unactivated PFL reveal that Gly-734 is buried 8 A from the surface of the protein in what we refer to here as the closed conformation of PFL. We provide here the first experimental evidence for an alternate open conformation of PFL in which: (i) the glycyl radical is significantly less stable; (ii) the activated enzyme exhibits lower catalytic activity; (iii) the glycyl radical undergoes less H/D exchange with solvent; and (iv) the T(m) of the protein is decreased. The evidence suggests that in the open conformation of PFL, the Gly-734 residue is located not in its buried position in the enzyme active site but rather in a more solvent-exposed location. Further, we find that the presence of the PFL-AE increases the proportion of PFL in the open conformation; this observation supports the idea that PFL-AE accesses Gly-734 for direct hydrogen atom abstraction by binding to the Gly-734 loop in the open conformation, thereby shifting the closed <--> open equilibrium of PFL to the right. Together, our results lead to a model in which PFL can exist in either a closed conformation, with Gly-734 buried in the active site of PFL and harboring a stable glycyl radical, or an open conformation, with Gly-734 more solvent-exposed and accessible to the PFL-AE active site. The equilibrium between these two conformations of PFL is modulated by the interaction with PFL-AE.