A liposomal preparation of glutathione (GSH) was investigated for its ability to replenish intracellular GSH and provide neuroprotection in an in vitro model of Parkinson's disease using paraquat plus maneb (PQMB) in rat mesencephalic cultures. In mixed neuronal/glial cultures depleted of intracellular GSH, repletion to control levels occurred over 4 h with liposomal-GSH or non-liposomal-GSH however, liposomal-GSH was 100-fold more potent; EC(50s) 4.75 μM and 533 μM for liposomal and non-liposomal-GSH, respectively. Liposomal-GSH utilization was also observed in neuronal cultures, but with a higher EC(50) (76.5 μM), suggesting that glia facilitate utilization. Blocking γ-glutamylcysteine synthetase with buthionine sulfoxamine prevented replenishment with liposomal-GSH demonstrating the requirement for catabolism and resynthesis. Repletion was significantly attenuated with endosomal inhibition implicating the endosomal system in utilization. Liposomal-GSH provided dose-dependent protection against PQMB with an EC(50) similar to that found for repletion. PQMB depleted intracellular GSH by 50%. Liposomal-GSH spared endogenous GSH during PQMB exposure, but did not require GSH biosynthesis for protection. No toxicity was observed with the liposomal preparation at 200-fold the EC(50) for repletion. These findings indicate that glutathione supplied in a liposomal formulation holds promise as a potential therapeutic for neuronal maintenance.