The Saccharomyces cerevisiae HOG pathway controls responses to osmotic shock such as production of the osmolyte glycerol. Here we show that the HOG pathway can be stimulated by addition of glycerol. This stimulation was strongly diminished in cells expressing an unregulated Fps1p glycerol channel, presumably because glycerol rapidly equilibrated across the plasma membrane. Ethanol, which passes the plasma membrane readily and causes water stress by disturbing the hydration of biomolecules, did not activate the HOG pathway. These observations suggest that stimulation of the HOG pathway is mediated by a turgor change and not by water stress per se.