The cellular localization of voltage-gated calcium channels (VGCCs) and synaptic vesicle-associated proteins, SV2, synapsin 1, and vesicle-associated membrane protein (VAMP) (synaptobrevin), was investigated in the guinea pig cochlea using immunocytochernistry and confocal laser scanning microscopy. Reactivity, in guinea pig, of antibodies to the alpha(1) subunits of L-type, alpha(1C) [Ca(nu)1.2] and alpha(1D) [Ca(v)1.3]; P/Q-type, alpha(1A)[Ca(nu)2.1]; and R-type, alpha(1E) [Ca(nu)2.3] high voltage-activated calcium channels, was determined by Western blotting and immunolabeling of cerebellum. In the cochlea the sensory inner hair cells of the organ of Corti displayed strong intracellular staining, predominantly localized to their basolateral poles, with an antibody directed against the oc,c subunit. Some alpha(1C) labeling was also observed in the inner pillar cells, in cell bodies of afferent neurons in the spiral ganglion, and in the inferior region of the spiral ligament. The supporting pillar cells were strongly immunoreactive throughout for alpha(1D), but no alpha(1D), labeling of the inner hair cells was seen. The alpha(1A), subunit showed a cytoplasmic distribution in all three rows of outer hair cells. alpha(1E) labeling localized to the outer hair cells, predominantly in the subcuticular plate region, and also to nerve fiber bundles beneath these hair cells. Strong immunoreactivity was consistently seen with antibodies directed against SV2 and synapsin I in neuronal structures surrounding the basolateral surfaces of both the inner and outer hair cells but was absent from the sensory cells themselves. VAMP labeling was found throughout the cytoplasm of the inner hair cells and in neuronal structures beneath the hair cells. These results reveal a differential distribution of VGCC-types in the sensory and nonsensory elements of the guinea pig cochlea, with the inner hair cells expressing alpha(1C)L-type channels and VAMP but not synapsin I or SV2.