The YOR163w open reading frame on chromosome XV of the Saccharomyces cerevisiae genome encodes a member of the MutT motif (nudix hydrolase) family of enzymes of M r 21,443. By cloning and expressing this gene in Escherichia coli and S. cerevisiae, we have shown the product to be a (di)adenosine polyphosphate hydrolase with a previously undescribed substrate specificity. Diadenosine 5,5ٟ-P 1 ,P 6 -hexaphosphate is the preferred substrate, and hydrolysis in H 2 18 O shows that ADP and adenosine 5-tetraphosphate are produced by attack at P  and AMP and adenosine 5-pentaphosphate are produced by attack at P ␣ with a K m of 56 M and k cat of 0.4 s ؊1 . Diadenosine 5,5ٟ-P 1 ,P 5 -pentaphosphate, adenosine 5-pentaphosphate, and adenosine 5-tetraphosphate are also substrates, but not diadenosine 5,5ٟ-P