Large conductance Ca2+-activated K+ channels (BKCa) encoded by the Slo1 gene play a role in the physiological regulation of many cell types. Here, we show that the beta1 subunit of Na+/K+-ATPase (NKbeta1) interacts with the cytoplasmic COOH-terminal region of Slo1 proteins. Reduced expression of endogenous NKbeta1 markedly inhibits evoked BKCa currents with no apparent effect on their gating. In addition, NKbeta1 down-regulated cells show decreased density of Slo1 subunits on the cell surface.