The dUDP analogue, 2'-deoxyuridine 5'-(alpha,beta-imido)diphosphate (dUPNP) was synthesized. The corresponding triphosphate analogue (dUPNPP) was prepared by enzymic phosphorylation of dUPNP using the enzyme pyruvate kinase and phosphoenolpyruvate as the phosphate donor. This method was successful in phosphorylating the imidodiphosphate analogue of 2'-deoxythymidine (dTPNP) to 2'-deoxythymidine 5'-(alpha, beta-imido)triphosphate (dTPNPP), in contradiction to a previous report. The properties of dUPNPP have been tested using the enzyme dUTPase from Escherichia coli. This enzyme, having a crucial role in nucleotide metabolism, is strictly specific for its substrate (dUTP) and catalyzes the hydrolysis of the alpha, beta-bridge, resulting in dUMP and pyrophosphate. Replacement of the alpha, beta-bridging oxygen in dUTP with an imido group resulted in a nonhydrolyzable substrate analogue and a potent competitive inhibitor of dUTPase (Ki = 5 microM). The analogue prepared (dUPNPP) may be utilized in crystallographic studies of the active site of dUTPase to provide knowledge about specific interactions involved in substrate binding and as a parental compound in design of dUTPase inhibition for medical purposes.