Mutants of Saccharomyces cerevisiae defective in the late steps of ergosterol biosynthesis are viable but accumulate structurally altered sterols within the plasma membrane. Despite the significance of pleiotropic abnormalities in the erg mutants, little is known about how sterol alterations mechanically affect the membrane structure and correlate with individual mutant phenotypes. Here we demonstrate that the membrane order and occurrence of voids are determinants of membrane rigidity and hypersensitivity to a drug. Among five ergDelta mutants, the erg2Delta mutant exhibited the most marked sensitivity to cycloheximide. Notably, measurement of time-resolved anisotropy indicated that the erg2Delta mutation decreased the membrane order parameter (S), and dramatically increased the rotational diffusion coefficient (D(w)) of 1-[4-(trimethylamino)pheny]-6-phenyl-1,3,5-hexatriene (TMA-DPH) in the plasma membrane by 8-fold, providing evidence for the requirement of ergosterol for membrane integrity. The IC(50) of cycloheximide was closely correlated with S/D(w) in these strains, suggesting that the membrane disorder and increasing occurrence of voids within the plasma membrane synergistically enhance passive diffusion of cycloheximide across the membrane. Exogenous ergosterol partially restored the membrane properties in the upc2-1erg2Delta strain. In this study, we describe the ability of ergosterol to adjust the dynamic properties of the plasma membrane, and consider the relevance of drug permeability.