The covalent structure of the pig kidney fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) subunit has been determined. Placement of the 335 amino acid residues in the polypeptide chain was based largely on automated Edman degradation of eight purified cyanogen bromide fragments generated from the S-carboxymethylated protein. The determination of the amino acid sequence of the largest cyanogen bromide fragment (154 residues) required additional analysis of subfragments obtained by tryptic cleavage at arginyl residues and by mild acid cleavage of an Asp-Pro peptide bond. Alignment of the cyanogen bromide fragments was accomplished by analysis of a product of limited proteolysis by an endogenous protease and by characterization of the tryptic peptides isolated from S-[14C]carboxymethylated fructose-1,6-bisphosphatase. This sequence information has permitted the identification of several reactive sites of functional and structural significance in pig kidney fructose-1,6-bisphosphatase.