Glutathione peroxidase-1 (GPX-1) is an enzyme that protects the lens against H2O2-mediated oxidative damage. The purpose of the present study was to determine the effects of GPX-1 knockout (KO) on lens transport and intracellular homeostasis. To investigate these lenses we used (1) whole lens impedance studies to measure membrane conductance, resting voltage and fiber cell gap junction coupling conductance; (2) osmotic swelling of fiber cell membrane vesicles to determine water permeability; and (3) injection of Fura2 and Na+-binding benzofuran isophthalate (SBFI) into fiber cells to measure [Ca2+]i and [Na+]i, respectively, in intact lenses. These approaches were used to compare wild-type (WT) and GPX-1 KO lenses from mice around 2 months of age. There were no significant differences in clarity, size, resting voltage, membrane conductance or fiber cell membrane water permeability between WT and GPX-1 KO lenses. However, in GPX-1 KO lenses, coupling conductance was 72% of normal in the outer shell of differentiating fibers and 45% of normal in the inner core of mature fibers. Quantitative Western blots showed that GPX-1 KO lenses had about 50% as much labeled Cx46 and Cx50 protein as WT, whereas they had equivalent labeled AQP0 protein as WT. Both Ca2+ and Na+ accumulated significantly in the core of GPX-1 KO lenses. In summary, the major effect on lens transport of GPX-1 KO was a reduction in gap junction coupling conductance. This reduction affected the lens normal circulation by causing [Na+]i and [Ca2+]i to increase, which could increase cataract susceptibility in GPX-1 KO lenses.