The intracellular functions of diadenosine polyphosphates are still poorly defined. To understand these better, we have expressed and characterized a heat stable, 16.6kDa Nudix hydrolase (Apf) that specifically metabolizes these nucleotides from a Drosophila melanogaster cDNA. Apf always produces an NTP product, with substrate preference depending on pH and divalent ion (Zn(2+) or Mg(2+)). For example, diadenosine tetraphosphate is hydrolysed to ATP and AMP with K(m), k(cat) and k(cat)/K(m) values 9microM, 43s(-1) and 4.8microM(-1)s(-1) (pH 6.5, 0.1mMZn(2+)) and 12microM, 13s(-1) and 1.1microM(-1)s(-1) (pH 7.5, 20mMMg(2+)), respectively. However, diadenosine hexaphosphate is efficiently hydrolysed to ATP only at pH 7.5 with 20mMMg(2+) (K(m), k(cat) and k(cat)/K(m) values of 15microM 4.0s(-1), and 0.27microM(-1)s(-1)). Fluoride potently inhibits diadenosine tetraphosphate hydrolysis in the presence of Mg(2+) (IC(50)=20microM), whereas it is ineffective in the presence of Zn(2+), supporting the view that inhibition involves a specific, MgF(3)(-)-containing transition state analogue complex. Patterns of Apf expression in Drosophila tissues show Apf mRNA levels to be highest in embryos and adult females. Subcellular localization with Apf-EGFP fusion constructs reveals Apf to be predominantly nuclear, having an apparent preferential association with euchromatin and facultative heterochromatin. This supports a nuclear function for diadenosine tetraphosphate. Our results show Apf to be a fairly typical member of the bis (5'-nucleosyl)-tetraphosphatase subfamily of Nudix hydrolases with features that distinguish it from a previously reported bis (5'-nucleosyl)-tetraphosphatase hydrolase activity from Drosophila embryos.