Site-specific disulfide cross-linking experiments detected a conformational change within the C-terminal segment of Escherichia coli pyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, upon substrate binding [Chang, Y.-Y., & Cronan, J. E., Jr. (1995) J. Biol. Chem. 270, 7896-7901]. The C-terminal lipid binding regions were cross-linked only in the presence of the substrate, pyruvate, and the thiamine pyrophosphate cofactor, indicating close proximity of a pair of C termini. We have now systematically substituted cysteine at 18 additional amino acid positions within the C-terminal region to obtain a panel of 21 proteins each having a single residue changed to cysteine. These proteins have been studied by disulfide cross-linking and by accessibility of the cysteine side chain to a variety of sulfhydryl agents. In the absence of pyruvate, the cysteine residues of the modified PoxB proteins failed to form disulfide bonds, generally failed to react with a large and rigid hydrophilic sulfhydryl reagent, 4-acetamido-4'-[(iodoacetyl)amino]stilbene-2,2'-disulfonic acid (IASD), and in some cases reacted weakly with a smaller more hydrophobic reagent, N-ethylmaleimide. Therefore, in this conformation, the C termini appear fixed in a rigid environment having limited exposure to solvent. In the presence of pyruvate, all of the C-terminal cysteine residues (except the two most distal from the C terminus) reacted with both sulfhydryl reagents and readily formed disulfide cross-linked species, indicating conversion to a structure having a high degree of conformational freedom. In the presence of lipid activators, Triton X-100 or dipalmitoylphosphatidylglycerol, a subset of the cysteine-substituted proteins no longer reacted with the membrane-impermeable IASD reagent, indicating penetration of these protein segments into the lipid micelles. For most of the proteins, similar extents of disulfide formation were seen upon addition of an oxidizing agent in the presence or absence of lipid activators. An exception was PoxB D560C which was much more readily cross-linked in the presence of lipid. Moreover, a subset of PoxB proteins that cross-linked to lower extents in the presence of lipids was found. The behavior of these proteins provides strong support for the model in which two C termini associate to form the functional lipid binding domain. These data are discussed in terms of three distinct PoxB conformers and the known crystal structure of a highly related protein.