In response to fluctuations in environmental osmolarity, yeast cells adjust their intracellular solute concentrations in order to maintain a constant turgor pressure and ensure continuation of cellular activity. In this study, the effect of hypo-osmotic stress on osmolyte content of osmotolerant yeasts Zygosaccharomyces rouxii and Pichia sorbitophila and the less tolerant Saccharomyes cerevisiae was investigated. All these yeasts released glycerol upon hypo-osmotic shock. However, Z. rouxii also released arabitol, whereas P. sorbitophila released erythritol in addition to arabitol and glycerol. Osmolyte release was very rapid and specific and was neither affected by reduced temperatures nor inhibited by the channel blocker gadolinium or the protonophore carbonyl cyanide m-chlorophenyl hydrazone. Extracellular osmolyte levels increased drastically suggesting that osmolytes were not metabolised but mainly released upon exposure to hypotonic conditions. The export process is well controlled, and the amount of osmolyte released was proportional to the shock intensity. Osmolyte release occurred with little cell lysis and thus the survival as well as the subsequent growth of yeast cells was largely unaffected after hypo-osmotic shock. The kinetics and patterns of osmolyte export suggest the involvement of channel proteins, but the molecular nature of this export pathway in yeasts, with exception of S. cerevisiae, remains to be established.