Following slow rates of cooling, at which cellular dehydration occurs, it is assumed that the intracellular environment vitrifies. However, the glass transition (Tg) temperature has not previously been determined for any cell type during cooling. Using differential scanning calorimetry (DSC), we have measured intracellularTg, in a range of cell types (bacteria, algae, yeasts) when cooled slowly in the presence of extracellular ice, where no cryoprotectants were added. Under the experimental conditions used, any intracellular freezing would have been detected by the release of latent heat associated with the change in entropy that accompanies the phase transition from liquid water to ice in the cell during cooling, or the reverse during warming; none was seen. Vitrification was detected by the variation in heat flow associated with the change in heat capacity, together with the absence of any release of latent heat. No vitrification signal was seen in control runs (medium without cells). The signal detected in the sample runs was the glass transition temperature of the maximally freeze-concentrated intracellular compartment (conventionally designated Tg’). The observed values of Tg’ ranged from just below −10 °C in thermophilic bacteria and the ice-nucleating bacterium Pseudomonas syringae, to around −25 °C in three species, including the snow alga Chlamydomonas nivalis. These temperatures are well above typical environmental temperatures for high latitudes in winter, indicating that intracellular vitrification must be a process of widespread ecological relevance for free-living single cells. These findings have a number of implications for cryopreservation: (a) During slow cooling, vitrification will occur at a high sub zero temperature and the cell will be osmotically unresponsive during subsequent cooling. On warming the vitrified cells will be exposed to an extracellular hypertonic stress before the cell interior become liquid. This set of physical events are not described in any current model of freezing injury, it is assumed that the cell is osmotically reactive at all temperatures down to the Tg of the extracellular solution. (b) Vitrification would be expected to stop any damaging intracellular events, but damage to the plasmalemma may still occur at the outer surface by direct exposure to hypertonic solutions. (c) Freeze drying of cells will always occur at a temperature below the intracellular vitrification temperature Source of funding: INRA, CEPIA Department, UMR 782 GMPA, F 78850 Thiverval Grignon, France. Conflict of interest: None declared. fonseca@grignon.inra.fr