Large conductance, Ca(2+)- and voltage-activated K(+) channels (BK) respond to two distinct physiological signals -- membrane voltage and cytosolic Ca(2+) (refs 1, 2). Channel opening is regulated by changes in Ca(2+) concentration spanning 0.5 micro M to 50 mM (refs 2-5), a range of Ca(2+) sensitivity unusual among Ca(2+)-regulated proteins. Although voltage regulation arises from mechanisms shared with other voltage-gated channels, the mechanisms of Ca(2+) regulation remain largely unknown. One potential Ca(2+)-regulatory site, termed the 'Ca(2+) 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 Ca(2+) bowl, accounts for all physiological regulation of BK channels by Ca(2+); the other site contributes to effects of millimolar divalent cations that may mediate physiological regulation by cytosolic Mg(2+) (refs 5, 13). Independent regulation by multiple sites explains the large concentration range over which BK channels are regulated by Ca(2+). This allows BK channels to serve a variety of physiological roles contingent on the Ca(2+) concentration to which the channels are exposed.