Using immunohistochemical and electrophysiological methods, we investigated the role of L-type Ca²⁺ channels in the regulation of the endocochlear potential (EP) of the endolymphatic surface cells (ESC) of the guinea pig stria vascularis. The following findings were made: (1) Administration of 30 μg/ml nifedipine via a vertebral artery significantly suppressed the transient asphyxia-induced decrease in the EP (TAID) and the transient asphyxia-induced increase in the Ca²⁺, referred to as TAIICa, concentration in the endolymph ([Ca]_e). (2) The endolymphatic administration of 1 μg/ml nifedipine significantly inhibited the TAID as well as the TAIICa. The endolymphatic administration of nifedipine (0.001–10 μg/ml) inhibited the TAID in a dose-dependent manner. (3) The endolymphatic administration of (+)-Bay K8644, an L-type Ca²⁺ channel closer, significantly inhibited the TAID, whereas (–)-Bay K8644, an L-type Ca²⁺ channel opener, caused a large decrease in the EP from ∼+75 mV to ∼+20 mV at 10 min after the endolymphatic administration. (4) By means of immunohistochemistry, a positive staining reaction with L-type Ca²⁺ channels was detected in the marginal cells of the stria vascularis. (5) Under the high [Ca]_e condition, we examined the mechanism of the TAIICa and hypothesized that the TAIICa might have been caused by the decrease in the EP through a shunt pathway in the ESC. (6) The administration of nifedipine to the endolymph significantly inhibited the Ba²⁺-induced decrease in the EP. These findings support the view that L-type Ca²⁺ channels in the marginal cells regulate the EP, but not directly the TAIICa.