The sulfhydryl groups of glycogen phosphorylase from rabbit muscle have been investigated with respect to their reactivity with p-chloromercuribenzoate (PCMB), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), cystine, N-ethylmaleimide (NEM), iodoacetic acid, and iodoacetamide. By spectrophotometric titration, PCMB reacts with 6 of the 18 half-cystine residues in native phosphorylase b, and with 12–14 of the 36 half-cystine residues in native phosphorylase a, and causes complete inactivation in both cases. Reaction of approximately 6 more residues in phosphorylase b (12 in phosphorylase a) occurs when the protein is denatured with HCl or sodium dodecyl sulfate (SDS).DTNB, cystine, and iodoacetic acid react with only two sulfhydryl groups in native phosphorylase b, and do not cause any loss of activity. Iodoacetamide can also react with two sulfhydryl groups without causing any loss of enzymic activity, but activity is lost in proportion to the extent of titration of the next four groups. DTNB reacts with 12 groups in SDS-denatured phosphorylase b but with only 9 groups in urea- or guanidine HCl-denatured enzyme. NEM and iodoacetic acid react with 14 groups in urea-denatured phosphorylase b. Two experiments are reported that provide additional confirmation of previous reports of the absence of disulfide bridges in phosphorylase.We conclude that two sulfhydryl groups per mole of phosphorylase b can react with any reagent without loss of enzymic activity. When the correct reagent and conditions are chosen, a further class of four sulfhydryl groups reacts, resulting in complete loss of enzymic activity. Thus, only two specific sulfhydryl groups on each monomer unit need be titrated to inactivate glycogen phosphorylase and cause it to dissociate. Upon denaturation of phosphorylase by several of the usual protein denaturants, another class of sulfhydryl groups reacts readily, the exact number depending on the reagent and the conditions, but the complete titration of all 18 sulfhydryl groups per molecule of phosphorylase b occurs only under special conditions. These results are of general relevance to procedures employing various protein denaturants and sulfhydryl reagents for the titration of protein sulfhydryl groups.