Aminoglycosides are widely used antibiotics and frequently produce acute ototoxicity. In this study we attempted to comparatively investigate the effects of gentamicin on Ca2+ influx of apical and basal outer hair cells (OHCs) isolated from guinea-pig cochlea. Since the solution of gentamicin sulfate salt is acidic (pH 3.1-3.3), we also explored the effect of external acidification on Ca2+ influx. By means of fura-2 microspectrofluorimetry, we measured the intracellular calcium concentration ([Ca2+]i) of OHCs bathed in Hanks' balanced salt solution (pH 7.40) during either a resting state or high K+-induced depolarization. Our results show that at the resting state, the baseline [Ca2+]i in apical OHCs (94+/-2.0 nM) was slightly lower than that in basal OHCs (101.1+/-2.4 nM). By contrast, the increase in [Ca2+]i evoked by high K+ depolarization in apical OHCs was about two-fold greater than that in basal OHCs. Nifedipine (30 microM) abolished the increased [Ca2+]i in both types of OHCs, suggesting that Ca2+ influx was mainly through L-type Ca2+ channels of OHCs. While gentamicin and extracellular acidification (pH 7.14) can separately attenuate this increase in [Ca2+]i in both types of OHCs, their suppressive effects are additive in basal OHCs, but not in apical OHCs. The implications of these findings are that: (1) apical and basal OHCs behave differently in response to depolarization-increased [Ca2+]i, and (2) basal OHCs are more vulnerable to the impairment of Ca2+ entry during depolarization by a combination of gentamicin and extracellular acidification, which is correlated with the clinical observation that ototoxicity of aminoglycosides at the basal coil of OHCs is more severe than that at the apical coils. Moreover, the possibility that extracellular acidification may enhance the acute ototoxic effects of aminoglycosides should be considered especially in topical applications.