Two genes from Caenorhabditis elegans and Saccharomyces cerevisiae, coding for enzymes homologous to the Nudix hydrolase family of nucleotide pyrophosphatases, have been cloned and expressed in Escherichia coli. The purified enzymes are homodimers of 39.1 and 43.5 kDa, respectively, are activated by Mg2+ and Mn2+, and are 30 to 50 times more active on NADH than on NAD(+). They both have a conserved array of amino acids downstream of the Nudix box first seen in the orthologous enzyme from E. coli which designates them as members of an NADH pyrophosphatase subfamily of the Nudix hydrolases.