Large-conductance Ca(2+)-activated potassium (BK) channels are composed of pore-forming alpha-subunits and auxiliary beta-subunits. The alpha-subunits are widely expressed in many cell types, whereas the beta-subunits are more tissue specific and influence diverse aspects of channel function. In the current study, we identified the presence of the smooth muscle-specific beta1-subunit in murine colonic tissue using Western blotting. The native beta1-subunits migrated in SDS-PAGE as two molecular mass bands. Enzymatic removal of N-linked glycosylations from the beta1-subunit resulted in a single band that migrated at a lower molecular mass than the native beta1-subunit bands, suggesting that the native beta1-subunit exists in either a core glycosylated or highly glycosylated form. We investigated the functional consequence of deglycosylating the beta1-subunit during inside-out single-channel recordings. During inside-out single-channel recordings, with N-glycosidase F in the pipette solution, the open probability (P(o)) and mean open time of BK channels increased in a time-dependent manner. Deglycosylation of BK channels did not affect the conductance but shifted the steady-state voltage of activation toward more positive potentials without affecting slope when Ca(2+) concentration was <1 microM. Treatment of myocytes lacking the beta1-subunits of the BK channel with N-glycosidase F had no effect. These data suggest that glycosylations on the beta1-subunit in smooth muscle cells can modify the biophysical properties of BK channels.