Abstract : The osmotic tolerance of microbial cells of different microorganisms was investigated as a function of glycerol concentration and temperatures. Cells displayed specific sensitivity to dehydration in glycerol solutions. The viability of Gram‐negative strains (Escherichia coli, Bradyrhizobium japonicum), Gram‐positive strains (Lactobacillus plantarum, L. bulgaricus), and yeasts (Saccharomyces cerevisiae, Candida utilis) decreased with increasing osmotic pressure. For each strain, a characteristic osmotic pressure threshold causing a loss of 40% of the population at the growth temperature was determined: 26–40 MPa for E. coli, 15–25 MPa for B. japonicum, 7–15 MPa for L. bulgaricus, 40–133 MPa for L. plantarum, 50–100 MPa for S. cerevisiae, and 15–26 MPa for C. utilis. Because this threshold varies with temperature, it was possible to construct a diagram that could be helpful to the determination of the sensitivity of each strain to osmotic stress as a function of osmotic pressure and temperature.