Rabbit muscle phosphofructokinase (E.C. 2.7.1.11; ATP: D-fructose-6-phosphate 1-phosphotransferase) consists of four protomers of 80 000 molecular weight, each of which contains 15--16 sulfhydryl groups. Specific cyanylation of the most reactive sulfhydryl group in each protomer with 2-nitro-5-thio-[14C]cyanobenzoic acid and cleavage of the S-cyanylated protomer yields two fragments--a 14C-labeled fragment of 72 500 molecular weight and an unlabeled fragment of 5400 molecular weight--indicating that the position of the cysteine residue bearing the most reactive sulfhydryl group is approx. 5400 daltons from the amino-terminal end of each protomer. The phosphofructokinase protomer also contains 5--6 sulfhydryl groups that are reactive in the denatured protomer but unreactive in the native protomer. The cleavage fragments, obtained from protomers specifically cyanylated at sulfhydryl groups reactive only in the denatured protomer, indicate that three of the cysteine residues bearing this class of sulfhydryl group occupy positions approx. 5000, 11 500, and 58 000 daltons from the carboxyl-terminal end of each protomer.