Bioreversible protection of the β-phosphate group of nucleoside diphosphates (NDPs) as bis(acyloxybenzyl)phosphate esters is presented. To investigate the structure-activity relationship of these potential NDP prodrugs (DiPPro drugs) a series of DiPPro compounds was synthesized bearing fatty acids of various lengths and d4T as a model nucleoside. For synthesis of the lipophilically modified diphosphate group, preformed phosphoramidites were allowed to react with nucleotides, and the β-P(III) moiety was subsequently oxidized. The chemical and enzymatic stability of these prodrugs was studied in different media such as phosphate buffer (pH 7.3) or CEM cell extracts. In all media, the hydrolysis rate was clearly dependent on the acyl moiety and decreased with increasing alkyl chain length. The compounds showed a markedly lower half-life in cell extracts than in pH 7.3 phosphate buffer due to the presence of enzyme-catalyzed cleavage. In all media, the DiPPro compounds released d4T diphosphate (d4TDP) as the main product beside d4TMP. In antiviral assays, the compounds proved to be at least as potent as d4T against HIV-1 and 2 in wild-type CEM/0 cells. As a proof of concept, compounds with longer acyl residues showed very good anti-HIV activities in thymidine-kinase-deficient cells (CEM/TK(-) ), indicating their ability to penetrate cell membranes and the delivery of phosphorylated metabolites.