The synthesis and in vitro anti-HIV activity of a novel series of pronucleotides are reported. These prodrugs were characterized by a phosphorodithiolate structure, incorporating two O-pivaloyl-2-oxyethyl substituents as biolabile phosphate protections. The compounds were obtained following an original one-pot three-step procedure, involving the formation of a phosphorodithioite intermediate which is in situ oxidized. In vitro, comparative anti-HIV evaluations demonstrate that such original prodrugs are able to allow the efficient intracellular release of the corresponding 5'-mononucleotide. The pronucleotide of 2',3'-dideoxyadenosine (ddA) 3 exhibited a very potent antiretroviral effect with 50% effective concentration (EC<sub>50</sub>) values in nanomolar concentration range in various cell lines. In primary monocytes/macrophages, this derivative was 500 times more potent in inhibiting HIV replication (EC<sub>50</sub> 0.23 pM) than ddA and the selectivity index of the prodrug is fifty times higher than the one of the parent nucleoside.