Nucleoside monophosphate kinases have an important role in the synthesis of nucleotides that are required for cellular metabolism. These enzymes are also important for the phosphorylation of nucleoside- and nucleotide analogs used in cancer and anti-viral therapy. We report the cDNA cloning and characterization of a 23 kDa guanylate kinase from Drosophila melanogaster (Dm-GUK). The predicted amino acid sequence was 58% identical to the human guanylate kinase and the enzyme was shown to phosphorylate GMP and dGMP with ATP as phosphate donor. The monophosphates of the deoxyguanosine analogs 2',2'-difluorodeoxyguanosine (dFdG) and 9-beta-D-arabinofuranosylguanine (araG) were also shown to be phosphorylated by the enzyme. We used the enzyme to reconstitute the complete in vitro three-step phosphorylation pathway for the conversion of dGuo and araG to the corresponding triphosphates.