BuPdGMPNHPP was synthesized and assayed as a non-incorporable inhibitor of B family DNA polymerases. The derivative was synthesized by preparation of the imidophosphorane of BuPdG followed by reaction with orthophosphate using the imidazolide method. BuPdGMPNHPP inhibited human DNA polymerase alpha and T4 DNA polymerase 10 and 3.5-times more potently than BuPdGTP, respectively, and was not a substrate for either enzyme. BuPdGMPNHPP acts as an active site affinity probe that could find use in co-crystallization trials of B family DNA polymerases.