ABSTRACT Yeast cells adapt to hyperosmotic shock by accumulating glycerol and altering expression of hundreds of genes. This transcriptional response of Saccharomyces cerevisiae to osmotic shock encompasses genes whose products are implicated in protection from oxidative damage. We addressed the question of whether osmotic shock caused oxidative stress. Osmotic shock did not result in the generation of detectable levels of reactive oxygen species (ROS). To preclude any generation of ROS, osmotic shock treatments were performed in anaerobic cultures. Global gene expression response profiles were compared by employing a novel two-dimensional cluster analysis. The transcriptional profiles following osmotic shock under anaerobic and aerobic conditions were qualitatively very similar. In particular, it appeared that expression of the oxidative stress genes was stimulated upon osmotic shock even if there was no apparent need for their function. Interestingly, cells adapted to osmotic shock much more rapidly under anaerobiosis, and the signaling as well as the transcriptional response was clearly attenuated under these conditions. This more rapid adaptation is due to an enhanced glycerol production capacity in anaerobic cells, which is caused by the need for glycerol production in redox balancing. Artificially enhanced glycerol production led to an attenuated response even under aerobic conditions. These observations demonstrate the crucial role of glycerol accumulation and turgor recovery in determining the period of osmotic shock-induced signaling and the profile of cellular adaptation to osmotic shock.