Reactivity of sulfhydryl groups in rabbit muscle pyruvate kinase toward 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) was studied in the presence of activating divalent metal ions, substrate, substrate analogue, and the allosteric inhibitor, L-Phe. The pattern of sulfhydryl modification in various complexes of pyruvate kinase was consistent with the extent of enzyme inactivation by DTNB under very similar conditions. The sulfhydryl reactivity of Mg(II)-, Co(II)-, and Mn(II)-substituted pyruvate kinase toward DTNB depended upon the nature of the activating divalent metal ions used in the following order of increasing potency, Mg(II) < Mn(II) < Co(II), which is inversely related to the order of catalytic efficiency of these metal ions at alkaline pH. Similar optical spectra and the patterns of sulfhydryl modification by DTNB of the metal derivatives of pyruvate kinase were observed upon the binding of the substrate, phosphoenolpyruvate (PEP), or the substrate analogue, phosphoglycolate, which also provided a complete protection against enzyme inactivation by DTNB. L-Phe, on the other hand, deprotected the enzyme from inactivation and further sulfhydryl modification by DTNB in the presence of PEP with the following order of potency depending upon the activating metal ions, Mn(II) < Co(II) < Mg(II), which parallels the order of metal dependency of L-Phe inhibition of this enzyme. L-Ala, which reverses the L-Phe inhibition of Mg(II)- or Co(II)-activated enzyme, restored the protective effect of PEP in the presence of L-Phe. The different patterns of sulfhydryl reactivity toward Mn(II)–enzyme (hyperbolic) and Mg(II)–enzyme (sigmoidal) correspond well with their kinetic patterns in the presence of L-Phe, indicating the presence of different conformational states between these two metal–enzyme complexes. These results led us to conclude that enzyme sulfhydryl reactivity toward DTNB can be used as a valid index for allosteric conformational changes of rabbit muscle pyruvate kinase.