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 0-mononucleotide. The pronucleotide of 2 0 ,3 0-dideoxyadenosine (ddA) 3 exhibited a very potent antiretroviral effect with 50% effective concentration (EC 50) 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 50 0.23 pM) than ddA and the selectivity index of the prodrug is fifty times higher than the one of the parent nucleoside.