BK_Ca channels are unique modulators of neuronal excitability that are activated by both membrane depolarization and elevation in intracellular Ca²⁺ concentration. In fact, channel activation under physiological conditions requires Ca²⁺ concentrations in the micromolar range as are known to occur in close spatial proximity of Ca²⁺-sources. Using affinity-purification and LC-MS/MS spectrometry on plasma membrane fractions from rat brain we show that BK_Ca channels are complexed with the voltage-gated calcium (Cav) channels Cav1.2 (L-type) and Cav2.1 (P/Q-type). In patch-clamp recordings on heterologously (CHO cells, Xenopus oocytes) expressed complexes (Cav a-subunits, a2[delta], Cavb, BKa, BKb4) Cav channels activated BK_Ca channels with characteristics identical to those described in native cells. The coupling between Cav and BK_Ca channels persisted in excised patches upon application of 10 mM EGTA, and required 10 mM of the fast Ca²⁺ chelator BAPTA to be disrupted. These results demonstrate that the 'micro-domain' organization suspected for activation of BK_Ca channels in many types of cell is realized by complexation of these channels with distinct Cav channel subtypes.