Calbindin-D28k is a member of the large EF-hand family of calcium-binding proteins, that is believed to function, in part as a cytosolic calcium buffer. Recent studies have demonstrated that cells containing Calbindin-D28k are protected from degeneration caused by conditions that elevate intracellular calcium concentrations. Since its initial discovery in 1966, Calbindin-D28k has been localized in the cytoplasm of many neuronal populations, but its nuclear localization has been uncertain. Using light and electron microscopic immunohistochemistry, and nuclear fractionation methods, we demonstrate localization of Calbindin-D28k not only in the cytoplasm, but also in the nucleus of rodent midbrain dopaminergic neurons and cerebellar Purkinje cells. The Calbindin-D28k immunoreactive staining intensity in the nucleus was routinely equal or greater than that in the cytoplasm. Since calcium signals are propagated to the nucleus, where they can regulate gene expression, the existence of nuclear Calbindin-D28k has important implications for cellular function.