Large conductance, Ca2+- and voltage-activated K+ channels (BK) respond to two distinct physiological signals - membrane voltage and cytosolic Ca2+ (refs 1, 2). Channel opening is regulated by changes in Ca2+ concentration spanning 0.5 μM to 50 mM (refs 2-5), a range of Ca2+ sensitivity unusual among Ca2+-regulated proteins. Although voltage regulation arises from mechanisms shared with other voltage-gated channels, the mechanisms of Ca2+ regulation remain largely unknown. One potential Ca2+-regulatory site, termed the 'Ca2+ bowl', has been located to the large cytosolic carboxy terminus. Here we show that a second region of the C terminus, the RCK domain (regulator of conductance for K+ (ref. 12)), contains residues that define two additional regulatory effects of divalent cations. One site, together with the Ca2+ bowl, accounts for all physiological regulation of BK channels by Ca2+; the other site contributes to effects of millimolar divalent cations that may mediate physiological regulation by cytosolic Mg2+ (refs 5, 13). Independent regulation by multiple sites explains the large concentration range over which BK channels are regulated by Ca2+. This allows BK channels to serve a variety of physiological roles contingent on the Ca2+ concentration to which the channels are exposed.