BK_Ca channels are fundamental in the control of cellular excitability (Lawson and McKay 2006). Thus, compounds that activate BK_Ca channels could provide potential therapies in the treatment of pathologies of the cardiovascular and central nervous system (Calderone 2002). We have previously demonstrated that a series of novel N-arylbenzamide compounds exhibit BK_Ca channel opening properties using an Rb⁺ efflux assay (Kirby R et al 2006). Using whole cell and excised patch clamp electrophysiology we have characterised the opener properties of BKOEt₁, an example of this chemical series, on a HEK293 cell line expressing the zero variant of the human BK_Ca alpha subunit. Whole-cell and inside-out patch clamp configurations were used. Currents were recorded using voltage step protocol (–100 to +100mV) in symmetrical potassium (140mM) with 0.3μM free Ca²⁺ in the bath and pipette solutions. Compounds were applied by continuous perfusion into the bath. Normalised conductance vs voltage (G-V) curves were plotted and V₅₀ values calculated. For single channel analysis currents were recorded for 60s at each voltage (-30mV to +80mV) from which open pore probability (NfPo) was calculated. In whole-cell configuration, BKOEt₁ (3, 10, 30 and 100μM) produced significant (p<0.001) leftward shifts in V₅₀ of 77.5±0.7mV, 43.9±6.3mV, 27.7±1.4mV and 15.9±0.4mV respectively (n=6-9) in G-V curve from control (V₅₀=90.1±2.0mV). The effects of BKOEt₁ (30μM) were abolished in the presence of the BK_Ca channel blocker paxilline (10μM). In inside-out configuration, BKOEt₁ (30μM) did not alter significantly the single-channel conductance (278.9±2.3pS, n=4) relative to control (267.1±3.3pS n=4). BKOEt₁ (10, 30 and 100μM) enhanced the activity of BK_Ca by increasing NfPo in a concentration-dependent manner as indicated by leftward shift in V₅₀ from control (79.5±0.4mV) of 40.1±1.7mV, 28.3±1.3mV, -3.9±0.9mV respectively (n=4-6). At +40mV the NfPo was significantly increased from 0.058±0.02 in control to 0.364±0.169, 0.813±0.098, and, 1.00±0.09 (n=4-6) in BKOEt₁ (10, 30 and, 100μM) respectively. The observed increase in NfPo by BKOEt₁ was abolished by paxilline (10μM). In conclusion, BKOEt₁ is a novel BK_Ca channel opener and that activation of the channel is brought about through increasing NfPo with no effect on single channel conductance.