The patch-clamp technique and fluorescence polarization analysis were used to study the dependence of Ca2(+)-dependent K+ channel kinetics and membrane fluidity on cholesterol (CHS) levels in the plasma membranes of cultured smooth muscle rabbit aortic cells. Mevinolin (MEV), a potent inhibitor of endogenous CHS biosynthesis was used to deplete the CHS content. Elevation of CHS concentration in the membrane was achieved using a CHS-enriching medium. Treatment of smooth muscle cells with MEV led to a nearly twofold increase in the rotational diffusion coefficient of DPH (D) and to about a ninefold elevation of probability of the channels being open (Po). The addition of CHS to the cells membrane resulted in a nearly twofold decrease in D and about a twofold decrease in Po. Elementary conductance of the channels did not change under these conditions. These data suggest that variations of the CHS content in the plasma membrane of smooth muscle cells affect the kinetic properties of Ca2(+)-dependent K+ channels presumably due to changes in plasma membrane fluidity. Our results give a possible explanation for the reported variability of Ca2(+)-dependent K+ channels kinetics in different preparations.