Cochlear outer hair cells have been well established as primary targets of the ototoxic actions of aminoglycoside antibiotics. These cells, isolated from the guinea pig cochlea and maintained in short-term culture, were used as a model for evaluating the acute effects of gentamicin on cell viability, depolarization-induced transmembrane calcium flux, and depolarization-induced motile responses. On the basis of morphology and fluorochromasia, the presence of extracellular gentamicin as high as 5 mM did not affect the viability of the cells for up to 6 hr, the longest time tested. Viable cells showed binding of fluorescently tagged gentamicin to their base but excluded the drug from their cytoplasm. In response to [K+]-depolarization, intracellular calcium levels (monitored with the fluorescent calcium-sensitive dye fluo-3) increased from a resting value of 218 +/- 102 nM to 2,018 +/- 1,077 nM concomitant with a cell shortening of 0.7% +/- 1.3%. The depolarization-induced calcium increase was apparently caused by calcium entry into the cell as it was inhibited by the calcium-channel blocker methoxyverapamil and prevented in the absence of extracellular calcium. Both gentamicin and neomycin blocked the [K+]-induced calcium increase at an IC50 of 50 microM. Despite the inhibition of calcium entry the ability of the outer hair cells to shorten under [K+]-depolarization was not impaired; in fact, cell shortening was even more pronounced in the absence of calcium influx (2.6% +/- 1.4%). This argues effectively against the existence of a calcium-dependent actomyosin-mediated component in [K+]-induced shape changes.(ABSTRACT TRUNCATED AT 250 WORDS)