A cDNA corresponding to the At3g26690 gene, which encodes a Nudix protein (AtNUDT13) with predicted mitochondrial localization, was isolated from an Arabidopsis thaliana library. The 202 amino acid AtNUDT13 polypeptide was overexpressed in Escherichia coli and purified to homogeneity. The preferred substrate for this hydrolase was diadenosine hexaphosphate (Ap(6)A), with K(m) and k(cat)/K(m) values of 0.61 mm and 16.0 x 10(3) m(-)1.s(-1), respectively. Optimal activity was at alkaline pH (8.5) with Mg(2+) (5 mm) as the cofactor. MS analysis revealed that the products of diadenosine hexaphosphate hydrolysis were ADP and adenosine tetraphosphate. Diadenosine pentaphosphate and adenosine tetraphosphate were additional substrates, but diadenosine tetraphosphate and diadenosine triphosphate, adenosine nucleotides, diphosphoinositol polyphosphate and phosphoribosyl pyrophosphate were not hydrolyzed. Chemical crosslinking and size exclusion chromatography demonstrated that the protein exists as a monomer in solution. Subcellular localization studies indicated that the AtNUDT13 protein is targeted to the mitochondria. This is the first description of a plant pyrophosphatase catalyzing the hydrolysis of long-chain diadenosine polyphosphates: molecules with multiple biological activities.