Plasma membrane integrity, ability to transport substrates and maintenance of homeostasis represent obligatory requirements for efficient ethanol production by Saccharomyces cerevisiae. The effect of ethanol on water diffusion through the bilayer and on mediated water movements was evaluated by stopped flow spectroscopy. Ethanol stimulated water diffusion and inhibited mediated water transport. In a strain overexpressing AQY1, the activation energy for water transport increased progressively (from 5.9 to 12.7 kcal mol-1) for increasing ethanol concentrations (up to 12% v/v), indicating that mediated water transport lost importance as compared with water diffusion through the bilayer. The effect of ethanol on proton movements (inward by passive diffusion and outward through the PMA1 H+-ATPase) was evaluated by measuring the rate of extracellular alcalinization and acidification of unbuffered cell suspensions at different temperatures. Above 10% ethanol, H+ diffusion was strongly increased at 30 degrees C, but no effect was observed at 20 degrees C up to 12%, indicating the existence of a threshold above which ethanol has a marked effect. On H+ extrusion, ethanol had no effect at 20 degrees C, but induced a monotonous decrease at higher temperatures. Our results support the view that above a threshold of ethanol concentration, the membrane structure is disrupted, becoming very leaky to H+.