A novel and convenient method for nucleoside triphosphate synthesis was applied to the preparation of potentially nonhydrolyzable xanthosine triphosphate derivatives. The N-methylimidazolide of xanthosine 5'-monophosphate reacted rapidly with methylenediphosphonic acid and imidodiphosphonic acid to give xanthosine 5'-(beta, gamma-methylene)triphosphate and xanthosine 5'-(beta, gamma-imido)triphosphate, respectively, in good yields. Both compounds inhibited the xanthosine-diphosphate-dependent prenylation of a mutant of Rab5, Rab5D136N, the nucleotide specificity of which had been converted from GTP to xanthosine triphosphate. The results indicate that xanthosine 5'-(beta, gamma-methylene)triphosphate and xanthosine 5'-(beta, gamma-imido)triphosphate bound to the mutant protein with similar affinities and were not hydrolyzed under the assay conditions. These novel derivatives may be useful tools for the study of the role of individual GTPases mutated to xanthosine triphosphate specificity in the background of other GTP-binding proteins.