AIM: 6-Chloropurines substituted at position 9 with bicyclic skeletons represent promising chemotherapeutic agents. We explored the metabolism and membrane transport of 9-norbornyl-6-chloropurine (NCP) aiming to understand its mechanism of action. MATERIALS AND METHODS: The metabolism of NCP was studied in vitro in whole cells (CCRF-CEM), cellular extracts, subcellular fractions and purified enzymes. Transport experiments were conducted in Caco-2 cell monolayers. RESULTS: Three metabolites were identified, a glutathione conjugate (NCP-GS), NCP-cysteinylglycine and NCP-cysteine. Both glutathione-S-transferase inhibition and glutathione (GSH) depletion prevented metabolite formation and increased the cytotoxicity of NCP. Transepithelial transport (Caco-2) indicated good permeability, with Papp (12.6±0.3) ×10(-5) cm/s. Importantly, the drug induced glutathione depletion in treated cells and affected the activity of several GSH-dependent enzymes. CONCLUSION: The novel nucleoside analog NCP represents a promising orally available antileukemic agent, acting through lowering of GSH levels in tumor cells.