Meristematic tissues from rye (Secale cereale) and oat (Avena sativa) were studied in an isothermal calorimeter at ؊3°C. When the frozen tissue was placed in the calorimeter, the pressure increased within 4 d to 25 and 9 kPa above ambient pressure in the sample vessels containing crowns of rye and oat, respectively. Concurrently, the thermal output went down to ؊194 W in rye over the 4-d period; this negative thermal activity could be accounted for by ice melting in the plants. When the pressure was released, the output from the calorimeter went from ؊194 to 229 W within 1 h, suggesting that water had frozen in the plants. We propose that CO 2 from respiration had dissolved in the water in the plants and caused melting of ice (heat absorption) due to the colligative properties of solutions. When the pressure was released, the CO 2 came out of solution and the water froze (heat evolution). These thermal observations were duplicated in a simplified, nonbiological system using a glycol/water mixture that was partially frozen at ؊3°C.Mechanisms used by plants to counter stresses during freezing are interactive and therefore are very difficult to characterize. Biochemical and biophysical adaptation of plants that occur at below-freezing temperatures but before injury occurs (second phase of hardening, 2PH) have been reported (Trunova, 1965;Steponkus, 1978;Olien, 1984;Livingston, 1996;Livingston and Henson, 1998). These adaptations conferred hardiness to 2PH plants by allowing them to survive stresses that plants hardened only at abovefreezing temperatures (first phase of hardening, 1PH) could not withstand (Olien, 1984).Ice formation in plants begins in the apoplast and must remain there if injury to the plant is to be avoided. Griffith et al. (1997) reported an increase in proteins with antifreeze activity in the apoplastic fluid from rye (Secale cereale) leaves that had been cold hardened at above-freezing temperatures. They suggested that these proteins provided protection down to freezing temperatures, and that other protective mechanisms allowed the plant to survive lower temperatures.Olien (1973, 1974, 1977, 1984) described a form of freezing stress called adhesion that occurs at around Ϫ10°C. Adhesion is the result of a slow rate of freezing such that very small displacements from equilibrium occur. Under these freezing conditions, the advancing ice lattice, upon reaching the vicinity of the cell wall, competes with it for the intervening liquid water; this competition causes adhesion between ice and the cell wall or between the cell wall and the plasmalemma. As the protoplast shrinks during freezing, adhesions to it can cause damage that results in the death of the plant (Olien, 1977).Adhesive stress may be relieved through the release of solutes, presumably from the hydrolysis of fructan (Olien, 1984). This would virtually convert adhesive stress to osmotic stress, as melting increases the amount of interfacial liquid. Solute release into the apoplast during 2PH was reported in rye, barley, and oat (Avena s...