Live outer hair cells were isolated from guinea pig, chinchilla, rat, mouse, and gerbil. The organ of Corti from selected turns of the cochlea was briefly incubated with collagenase and outer hair cells were separated from the tissue by micromanipulation under microscopic observation.Morphological criteria for cell viability were: (1) cylindrical cell shape without swelling or distortion of the membrane;(2) location of the nucleus in its normal position near the base of the cell; (3) cytoplasm devoid of Brownian motion and granulation.Both yield and quality (as judged by these morphological criteria) of isolated hair cells varied with the species and the turn from which the isolation was attempted. Consistently high yields and cells of good morphology were obtained from guinea pigs and chinchillas.Fewer cells were obtained from rats and mice, and their quality was less consistent. Gerbils gave the poorest yield and quality of outer hair cells. In all species, the preparation was more successful from the apical than from the basal turn. The length of apical hair cells varied almost 4-fold from 60 to 80 pm in guinea pig and chinchilla to 20 to 40 pm in the other species, while their diameter only varied 1.5-fold from 7 (mouse) to 10 pm (chinchilla). Outer hair cells could be maintained in vitro in good condition for several hours. Typical early signs of degeneration were increased Brownian motion and granulation in the cytoplasm, upward movement of the nucleus, or distortion of cell shape. Degeneration was always accompanied by a shortening along the long axis.