Diphosphate-modified substrates for prenyltransferase were synthesized and examined as substrates for the prenyltransferase reaction. They were dimethylallyl methylenediphosphonate, geranyl methylenediphosphonate, geranyl imidodiphosphate, geranyl phosphosulfate, farnesyl methylenediphosphonate, farnesyl imidodiphosphate, and farnesyl phosphosulfate. All of them except dimethylallyl methylenediphosphonate were accepted as substrates by solanesyl diphosphate synthase to give solanesyl diphosphate and the former four analogs were also accepted as substrates by farnesyl diphosphate synthase to give farnesyl diphosphate. The Km values of both enzymes for the methylenediphosphonate and imidodiphosphate analogs were comparable to those of the corresponding diphosphate substrates, but the phosphosulfate analogs showed much greater Km values than the diphosphate substrates. On the other hand, the Vmax values for these artificial substrates were all smaller than those for the corresponding natural substrates. Kinetic experiments with the analogs showed that the ionization-condensation-elimination mechanism proposed for the farnesyl diphosphate synthase reaction holds also for the solanesyl diphosphate synthase reaction and that the diphosphoryl structure, capable of chelating with divalent cations, is important topologically and kinetically rather than thermodynamically.