Large conductance Ca(2+)-activated potassium channels (BK(Ca) channels) are expressed in the plasma membrane of various cell types. Interestingly, recent studies provided evidence for the existence of BK(Ca) channels also in mitochondria. However, the molecular composition of these channels as well as their cellular and tissue distribution is still unknown. The goal of the present study was to find a candidate for the regulatory component of the mitochondrial large conductance calcium activated potassium (mitoBK(Ca)) channel in neurons. A combined approach of Western blot analysis, high-resolution immunofluorescence and immunoelectron microscopy with the use of antibodies directed against four distinct beta subunits demonstrated the presence of the BK(Ca) channel beta4 subunit (KCNMB4) in the inner membrane of neuronal mitochondria in the rat brain and cultured neurons. Within the cell, the expression of beta4 subunit was restricted to a subpopulation of mitochondria. The analysis of beta4 subunit distribution throughout the brain revealed that the highest expression levels occur in the thalamus and the brainstem. Our results suggest that beta4 subunit is a regulatory component of mitochondrial BK(Ca) channels in neurons. These findings may support the perspectives for the neuroprotective role of mitochondrial BK(Ca) channel in specific brain structures.