Exposure of cultured bovine lens epithelial cells (BLECs) to minimal essential medium (MEM) containing 40 mM galactose (Gal) promotes a decrease in the cellular content of reduced glutathione (GSH) as galactitol increases. Incubation of BLECs with Gal also leads to a reduction in 3H-myo-inositol (3H-MI) concentrating capability. Studies were therefore initiated to determine the nature of the relationship between polyol accumulation, GSH content and the attenuation of the active transport of myo-inositol. The inhibitor of glutathione biosynthesis, L-buthionine sulfoximine (L-BSO) was used in order to lower the intracellular pool of GSH in MEM-maintained cells to a concentration below that characteristically observed in Gal-treated cells, under conditions whereby no galactitol accumulation could or had occurred. L-BSO (0.5 mM) was simultaneously administered to BLECs maintained in either MEM or Gal for up to five days. The cellular content of GSH after five days of continuous incubation was 3.3 μg GSH/μg PO4 in MEM alone and 0.45 μg GSH/μg PO4 in MEM + BSO. Moreover, the GSH content in BLECs exposed to Gal for five days was 1.9 μg GSH/μg PO4 and was not detectable in the Gal + BSO-treated cells. However, the ability to concentrate 3H-MI in MEM + BSO-treated BLECs was equivalent to that observed with MEM-maintained cells regardless of the significant difference in GSH content. Likewise, L-BSO addition to Gal-treated cells, while virtually depleting the intracellular GSH content, did not further decrease the ability of the cells to accumulate H-MI compared to that observed with BLECs in Gal alone. Indeed, supplementation of Gal-treated cells with exogenous GSH failed to correct the Gal-induced attenuation in myo-inositol concentrating ability. These studies demonstrate that the Gal-induced depletion of cellular GSH and the Gal-induced deficit in ability to concentrate myoinositol are not associated and represent independent events. That is, depletion of lens cell GSH does not lead to the attenuation of myo-inositol uptake in cultured lens epithelial cells.