Stimulation of A-431 carcinoma cells with epidermal growth factor (EGF) causes dramatic morphologic responses including ruffling, rounding, and bulk-phase pinocytosis. In attempts to explore the mechanisms responsible for changes in plasmalemma topography, we have investigated the effects of exogenous sterols thought to alter membrane fluidity. Light and scanning electron microscopy revealed a time- and concentration-dependent inhibition of ruffling (greater than 90%) by cholesterol. This effect could be duplicated by preincubation of the cells with comparable levels of low-density lipoproteins (LDL). EGF-stimulated bulk-phase endocytosis also is inhibited by treatment with cholesterol. No alteration of EGF binding, kinase stimulation, or internalization was detected in cells incubated in cholesterol-enriched medium (175 micrograms/ml in 0.5% ethanol), nor did cholesterol or LDL have any effect on EGF-stimulated rounding. Morphometry of electron micrographs from cholesterol-treated cells revealed a selective depletion of interdigitating lateral surface membrane that normally appears to be recruited to generate apical ruffles. Thus, the sterol inhibition of ruffling may be due to redistribution of plasmalemma rather than to changes in membrane viscosity. Together with previous observations, these data suggest that EGF-stimulated ruffling and bulk-phase pinocytosis are related phenomena, whereas EGF-stimulated cell rounding is an independent process.