Glutathione (GSH) is a negatively charged tripeptide, which is a major determinant of the cellular redox state and defense against oxidative stress. It is assembled inside and degraded outside the cells and is released under various physiological and pathophysiological conditions. The GSH release mechanism is poorly understood at present. In our experiments, freshly isolated rat thymocytes were found to release GSH under normal isotonic conditions at a low rate of 0.82±0.07 attomol/cell/min and that was greatly enhanced under hypoosomotic stimulation to reach a level of 6.1±0.4 attomol/cell/min. The swelling-induced GSH release was proportional to the cell density in the suspension and was temperature-dependent with relatively low activation energy of 5.4±0.6 kcal/mol indicating a predominant diffusion mechanism of GSH translocation. The osmosensitive release of GSH was significantly inhibited by blockers of volume-sensitive outwardly rectifying (VSOR) anion channel, DCPIB and phloretin. In patch-clamp experiments, osmotic swelling activated large anionic conductance with the VSOR channel phenotype. Anion replacement studies suggested that the thymic VSOR anion channel is permeable to GSH(-) with the permeability ratio P(GSH)/P(Cl) of 0.32 for influx and 0.10 for efflux of GSH. The osmosensitive GSH release was trans-stimulated by SLCO/OATP substrates, probenecid, taurocholic acid and estrone sulfate, and inhibited by an SLC22A/OAT blocker, p-aminohippuric acid (PAH). The inhibition by PAH was additive to the effect of DCPIB or phloretin implying that PAH and DCPIB/phloretin affected separate pathways. We suggest that the VSOR anion channel constitutes a major part of the γ-glutamyl cycle in thymocytes and, in cooperation with OATP-like and OAT-like transporters, provides a pathway for the GSH efflux from osmotically swollen cells.