Saccharomyces cerevisiae NCYC 366 is susceptible to cold osmotic shock. Exponentially growing cells from batch cultures grown in defined medium at 30 C, after being suspended in 0.8 m mannitol containing 10 mm ethylenedia-minetetraacetic acid and then resuspended in ice-cold 0.5 mm MgCl₂, accumulated the nonmetabolizable solutes d-glucosamine-hydrochloride and 2-aminoisobutyrate at slower rates than unshocked cells; shocked cells retained their viability. Storage of unshocked batch-grown cells in buffer at 10 C led to an increase in ability to accumulate glucosamine, and further experiments were confined to cells grown in a chemostat under conditions of glucose limitation, thereby obviating the need for storing cells before use. A study was made of the effect of the different stages in the cold osmotic shock procedure, including the osmotic stress, the chelating agent, and the cold Mg²⁺-containing diluent, on viability and solute-accumulating ability. Growth of shocked cells in defined medium resembled that of unshocked cells; however, in malt extract-yeast extract-glucose-peptone medium, the shocked cells had a longer lag phase of growth and initially grew at a slower rate. Cold osmotic shock caused the release of low-molecular-weight compounds and about 6 to 8% of the cell protein. Neither the cell envelope enzymes, invertase, acid phosphatase and l-leucine-β-naphthylamidase, nor the cytoplasmic enzyme, alkaline phosphatase, were released when yeast cells were subjected to cold osmotic shock.