BK_Ca channels are fundamental in the control of cellular excitability and have been identified as potential therapeutic targets in many pathological conditions (Lawson and McKay 2006). We have previously optimised a non-radioactive Rb⁺ efflux assay for the assessment of BK_Ca channel activity (Kirby R et al 2006). This assay was used to characterise a series of novel N-arylbenzamide compounds for BK_Ca channel opener activity (Calderone V, 2006) using a recombinant HEK293 cell line expressing either the human α-subunit or co-expression with the β1-subunit of BK_Ca channel. Cells were incubated in RbCl (5.4mM) buffer for 4 hours then washed prior to incubation in (5.4mM) KCl buffer containing either test compound (0.1-100μM) or vehicle (DMSO, 0.3%) for 10 minutes. The supernatant and lysate samples were collected and the Rb⁺ content was determined by atomic absorption spectrometry and Rb⁺ efflux (%) was calculated. Data are presented as mean values (±SEM). All novel compounds tested on the BK_Ca channel α-subunit cell line evoked concentration related increases in Rb⁺ efflux. The maximum increase in Rb⁺ efflux (Emax) evoked by N-aryl-benzamide compounds ranged between 53-91% (n=8) above that the baseline of 22.2±1.1% (n=10-12); that achieved with NS1619 (reference BK_Ca channel opener) was 62.5±4.8% (n=8). The series of compounds demonstrated a 40-fold potency range (EC₄₀: 0.12-2.95μM); EC₄₀ for NS1619 was 0.35±0.40μM (n=8). In the presence of the BK_Ca channel blocker, iberiotoxin (0.1μM), the compounds (100μM for 10 minutes) failed to increase Rb⁺ efflux. All compounds tested on the BK_Ca channel α- and β1 subunit evoked significant concentration related increases in Rb⁺ efflux. The Emax for the compounds was in the range of 50-87%, and that achieved for NS1619 was 65.3±2.7% (n=8). The compounds demonstrated a 220-fold range in potency (EC₄₀: 0.44-96.0µM) and that for NS1619 was 0.68±0.37µM (n=8). The increase in Rb⁺ efflux due to the compounds (100μM) in cells expressing the α- and β1 subunit was abolished by Iberiotoxin (0.1μM). In conclusion, the N-aryl-benzamide compounds tested exhibit properties consistent with the activation of BK_Ca channels. Preliminary data indicate discrimination, based on efficacy and potency, to activate BK_Ca channels composed of α-subunit only or in the presence of β1 subunit by this series of compounds. Such agents will assist in the development of pharmacophore models to give insight into the receptor site for openers within the BK_Ca channel protein.