Carbon monoxide (CO) is a potent activator of large conductance, calcium-dependent potassium (BK Ca) channels of vascular myocytes and carotid body glomus cells or when heterologously expressed. Using the human BK Ca channel alpha1-subunit (hSlo1; KCNMA1) stably and transiently expressed in human embryonic kidney 293 cells, the mechanism and structural basis of channel activation by CO was investigated in inside-out, excised membrane patches. Activation by CO was concentration dependent (EC50 approximately 20 microM), rapid, reversible, and evoked a shift in the V 0.5 of -20 mV. CO evoked no changes in either single channel conductance or in deactivation rate but augmented channel activation rate. Activation was independent of the redox state of the channel, or associated compounds/protein partners, and was partially dependent on [Ca2+]i in the physiological range (100-1,000 nM). Importantly, CO "super-stimulated" BK Ca activity even in saturating [Ca2+]i. Single or double mutation of two histidine residues previously implicated in CO sensing did not suppress CO activation but replacing the S9-S10 module of the C-terminal of Slo1 with that of Slo3 completely prevented the action of CO. These findings show that a motif in the S9-S10 part of the C-terminal is essential for CO activation and suggest that this gas transmitter activates the BK Ca channel by redox-independent changes in gating.