Three members of the Nudix hydrolase superfamily (NUcleoside DIphosphate X) have been cloned from Escherichia coli MG1655 and expressed. The proteins have been purified and identified as enzymes active on nucleoside diphosphate derivatives with the following specificities: Orf141 (yfaO) is a nucleoside triphosphatase preferring pyrimidine deoxynucleoside triphos-phates. Orf153 (ymfB) is a non-specific nucleoside tri-and diphosphatase, and atypically releases inorganic ortho-phosphate from triphosphates instead of pyrophosphate. Orf191 (yffH) is a highly active GDP-mannose pyrophos-phatase. All three enzymes require a divalent cation for activity and are optimally active at alkaline pH, characteristic of the Nudix hydrolase superfamily. The question of whether or not Orf1.9 (wcaH) is a bona fide member of the Nudix hydrolase superfamily is discussed.