The effect of glutathione co-therapy on the expression of gentamicin ototoxicity was tested in pigmented guinea pigs. The first group of animals was injected with gentamicin (100 mg/kg body weight/day) for two weeks followed by 10 weeks of rest. A second group received glutathione by gastric gavage immediately prior to each gentamicin injection. Two groups of controls were treated either with saline injections or glutathione gavage alone. Auditory brainstem responses, taken at 2-week intervals, revealed a progressive gentamicin-induced hearing loss reaching a 30 to 40 dB threshold shift at 2 kHz, approximately 60 dB at 8 kHz and 80 dB at 18 kHz. Glutathione co-therapy slowed the progression of hearing loss and significantly attenuated the final threshold shifts by 20 to 40 dB. Morphological evaluation confirmed hair cell loss after gentamicin treatment and protection by glutathione. Drug serum levels were assayed at 2 and 7 days of treatment. There were no differences between the gentamicin (mean = 183 micrograms/ml; range, 90 to 300) and the gentamicin/glutathione group (mean = 164 micrograms/ml; range, 80 to 320). Antimicrobial activity of gentamicin was tested against Staphylococcus aureus and Pseudomonas aeruginosa. A 30-fold molar excess of glutathione did not influence the efficacy of gentamicin. These studies suggest that glutathione protects against ototoxicity by interfering with the cytotoxic mechanism.