The comparative ototoxicity of hydroxygentamicin (Win 42,122-2), a new aminoglycosidic antibiotic, gentamicin and kanamycin was evaluated in guinea pigs by assessment of the Preyer (pinna) reflex response to pure tone frequencies ranging from 2.5 to 20.0 KHz, and by histologic examination of surface preparations of the organ of Corti. Daily subcutaneous administration of 80.0 mg/kg of gentamicin or 240.0 mg/kg of kanamycin to groups of six guinea pigs for 18 to 45 days resulted in loss of the Preyer reflex in all animals. The Preyer reflex was retained in 5 of 6 guinea pigs given 80.0 mg/kg/day of hydroxygentamicin for 77 days and in 6 of 6 guinea pigs given 160.0 mg/kg/day for the same period. Microscopic examination of cochleas from guinea pigs given gentamicin or kanamycin revealed extensive outer and inner hair cell loss in all animals. Cytocochleograms of 5 of 6 guinea pigs medicated with 160.0 mg/kg of hydroxygentamicin were comparable to those of the controls. In the sixth guinea pig there was a localized lesion involving all three rows of outer hair cells and some inner hair cells in the second turn. The results of this study indicated that hydroxygentamicin may be tolerated better than gentamicin in the guinea pig and therefore warrants further development as a new and less toxic aminoglycosidic antibiotic.