The present study has resulted in the development of a procedure for the specific chemical fragmentation of human phosphoglucose isomerase into a minimal number of peptides. A two-cycle procedure for cleaving the protein with 2-nitro-5- thiocyanobenzoic acid results in four primary peptides and three overlap peptides. The peptides can be readily separated on the basis of their size by using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Preliminary peptide alignments have been considered, and amino acid analyses have been performed. End-terminal analyses of the enzyme revealed a carboxyl terminal sequence of Asp-Val-Gln and a blocked amino terminus. The cysteine cleavage procedure provides an excellent method for the identification and location of specific genetic mutations of human phosphoglucose isomerase.