Biochemical and metabolic transformations of 3′-azido-3′- deoxythymidine 5′-choline phosphate (1) were studied using its32P-labelled counterpart for the evaluation of possible reasons for its enhanced anti-HIV activity. An effective synthesis of32P- labelled 1 with a specific activity 1,000 Ci/mmol was developed by esterification of32P-phosphoric acid with choline in the presence of BrCN followed by the coupling of the resulting choline phosphate with 3′-azido-3′-deoxythymidine (AZT). Chemical and enzymatic stabilities of 1 as well as the dynamics of penetration through HL-60 cell membranes were studied at the concentrations comparable to its antiviral concentrations. The products of intracellular transformations of the studied nucleotide were identified. Copyright ©Taylor & Francis Group, LLC.