Large conductance calcium activated potassium channels (BKCa) are fundamental in the control of cellular excitability. Thus, compounds that activate BKCa channels could provide potential therapies in the treatment of pathologies of the cardiovascular and central nervous system. A series of novel N-arylbenzamide compounds, and the reference compound NS1619, were evaluated for BKCa channel opener properties in Human Embryonic Kidney (HEK293) cells expressing the human BKCa channel α-subunit alone or α+β1-subunit complex. Channel activity was determined using a non-radioactive Rb(+) efflux assay to construct concentration effect curves for each compound. All N-arylbenzamide compounds and NS1619 evoked significant (p <0.05) concentration related increases in Rb(+) efflux both in cells expressing α-subunit alone or α+β1-subunits. Co-expression of the β1-subunit modified the Rb(+) efflux responses, relative to that obtained in cells expressing the α-subunit alone, for most of the N-arylbenzamide compounds, in contrast to NS1619. The EC40 values of NS1619, BKMe1 and BKOEt1 were not significantly affected by the co-expression of the BKCa channel α+β1-subunits. In contrast, 5 other N-arylbenzamides (BKPr2, BKPr3, BKPr4, BKH1 and BKVV) showed a significant (p <0.05) 2- to 10-fold increase in EC40 values when tested on the BKCa α+β1-subunit expressing cells compared to BKCa α-subunit expressing cells. Further, the Emax values for BKPr4, BKVV and BKH1 were lower in the BKCa channel α+β1-subunit expressing cells. In conclusion, the N-arylbenzamides studied, like NS1619, were able to activate BKCa channels formed of the α-subunit only. The co-expression of the β1-subunit, however, modified the ability of certain compounds to active the channel leading to differentiated pharmacodynamic profiles.