Physiological evidence has shown that cochlear outer hair cells (OHC) possess L-type voltage-dependent Ca2+ channels through which Ca2+ enters the OHC during depolarization. Their subcellular distribution has, however, remained unclear. In this study, the distribution of L-type Ca2+ channels on the basolateral plasma membrane of OHC has been demonstrated by the use of a laser scanning confocal microscope (LSCM) and a fluorescent probe DMBODIPY-DHP. The fluorescent staining pattern on the basolateral wall is nonuniform, suggesting a heterogeneous distribution of the channels in the plasma membrane. Direct imaging of intracellular Ca2+ visualized in real time by means of the LSCM and the fluorescent Ca2+ probe fluo 3 revealed temporal and spatial integration of Ca2+ movements and Ca2+ channel distribution. Exposure to high-K+ solution induced heterogeneity in the subcellular increase in the intracellular Ca2+ concentration. These results suggest that the heterogeneous distribution of L-type Ca2+ channels on the basolateral membrane might induce heterogeneous intracellular Ca2+ distribution during electrical activity in the OHC.