Carbon monoxide (CO) is a potent activator of large conductance, calcium-dependent potassium (BK_Ca) channels when heterologously expressed in HEK293 cells (Williams et al., 2004; Jaggar et al., 2005) or when natively expressed in carotid body glomus (Riesco-Fagundo et al., 2001) and vascular smooth muscle cells (Jaggar et al., 2002). Cellular CO production emanates primarily from the enzymatic catalysis of heme by hemeoxygenases, a process which has the strict co-requirement for nicotinamide adenine dinucleotide phosphate (NADPH) and molecular oxygen (O₂). Hemeoxygenase-2 (HO-2) is a protein partner closely associated with the BK_Ca channel complex and co-application of its substrates, NADPH, heme and O₂, evoke channel activation which is rapidly reversed upon removal of O₂ (Williams et al., 2004). Such data suggest that HO-2 functions as an O₂ sensor in tissues where BK_Ca and HO-2 are co-expressed. Here, using the BK_Ca channel α-subunit (KCNMA1 – BKα) stably expressed in HEK293 cells, we investigated the mechanism of channel activation by CO in inside-out excised membrane patches. CO was introduced into the bathing solution by using the well-characterised chemical CO-donor, tricarbonyldichlororuthenium (II) dimer ([Ru(CO₃)Cl₂]); the breakdown product of this donor was utilised as a control. CO evoked a rapid activation of BKα which was concentration-dependent (EC₅₀ = 37.9 ± 4.3 µM, mean ± S.E.M., 35 observations from 7 patches) and freely reversible. Single channel conductance (in symmetrical 140 mM potassium solutions, [Ca²⁺]_i = 336 nM) was unaltered upon treatment with CO (187.0 ± 4.8 pS vs. 173.9 ± 6.9 pS (n = 7, P > 0.1, Student’s paired t-test)). However, CO evoked an increase in the rate of channel activation which was reflected in a reduction in the activation time constants from, for example, 11.82 ± 3.70 ms to 3.7 ± 0.95 ms (at +50 mV, n = 7, P < 0.01) without affecting the deactivation time constants. CO activation demonstrated a dependence on [Ca²⁺]_i with a window of between 100 nM and 10 µM. Importantly, application of CO within this Ca²⁺ window was able to superstimulate BKα activity to levels not achieved by saturating [Ca²⁺]_i. These data show that CO robustly stimulates BK_Ca channels principally by increasing the rate of channel opening. The β subunit is not necessary for this regulatory event. Although a pivotal event in this CO-evoked stimulation is clearly an increase in the intrinsic Ca²⁺-sensitivity of the α-subunit, a major component of this activation occurs independently of Ca²⁺, via a hitherto unidentified mechanism.