The product of the Escherichia coli orf1.9, or yefc, gene (GenBank accession number L11721) has been expressed under the control of a T7 promoter, purified to apparent homogeneity, and identified as a novel enzyme that hydrolyzes GDP-mannose or GDP-glucose to GDP and the respective hexose. The enzyme has little or no activity on other nucleotides, dinucleotides, nucleotide sugars, or sugar phosphates. It has a pH optimum between 9.0 and 9.5, a Km of 0.3 mM, and a Vmax of 1.6 mumol min-1 mg-1 for GDP-mannose, and it requires divalent cations for activity. This enzyme of 160 amino acids (M(r) = 18, 405) contains the consensus sequence GX(I/L/V)(E/Q)(X)2ET(X)6R(X)4E(X)2(I/L), characteristic of the MutT family of proteins and previously shown to form part of the nucleotide-binding site of MutT (Frick, D. N., Weber, D. J., Abeygunawardana, C., Gittis, A. G., Bessman, M. J., and Mildvan, A. S. (1995) Biochemistry 34, 5577-5586). A comparison of the enzymatic reactions catalyzed by the GDP-mannose mannosyl hydrolase and the other enzymes of the MutT family suggests that the consensus signature sequence designates a novel nucleoside diphosphate binding site and catalytic motif.