The generation of reactive oxygen species (ROS) is thought to be part of the mechanism underlying noise-induced hearing loss (NIHL). Glutathione (GSH) is an important cellular antioxidant that limits cell damage by ROS. In this study, we investigated the effectiveness of a GSH supplement to protect GSH-deficient animals from NIHL. Pigmented guinea pigs were exposed to a 4 kHz octave band noise, 115 dB SPL, for 5 h. Group 1 had a normal diet, while groups 2, 3 and 4 were fed a 7% low protein diet (leading to lowered tissue levels of GSH) for 10 days prior to noise exposure. One hour before, immediately after and 5 h after noise exposure, subjects received either an intraperitoneal injection of 5 ml/kg body weight of 0.9% NaCl (groups 1 and 2), 0.4 M glutathione monoethyl ester (GSHE; group 3) or 0.8 M GSHE (group 4). Auditory thresholds were measured by evoked brain stem response at 2, 4, 8, 12, 16 and 20 kHz before and after noise exposure. Ten days post exposure, group 1 showed noise-induced threshold shifts of approximately 20 dB at 2, 16 and 20 kHz and 35 to 40 dB at other frequencies. Threshold shifts in group 2 were significantly greater than baseline at 2, 4, 16 and 20 kHz. GSHE supplementation in a dose-dependent fashion attenuated the threshold shifts in the low protein diet animals. Hair cell loss, as evaluated with cytocochleograms, was consistent with the auditory-evoked brainstem response results. Group 2 exhibited significantly more hair cell loss than any of the other groups; hair cell loss in group 3 was similar to that seen in group 1; group 4 showed less loss than group 1. These results indicate that GSH is a significant factor in limiting noise-induced cochlear damage. This is compatible with the notion that ROS generation plays a role in NIHL and that antioxidant treatment may be an effective prophylactic intervention.