Protein crystals are usually grown in multi-component aqueous solutions containing salts, buffers and other additives. To measure the X-ray diffraction data of the crystal, crystals are rapidly lowered to cryogenic temperatures. On flash cooling, ice frequently forms affecting the integrity of the sample. In order to eliminate this effect, substances called cryoprotectants are added to produce a glassy (vitrified) state rather than ice. Heretofore, the quantity of cryoprotectant needed to vitrify the sample has largely been established by trial and error. In this study, differential scanning calorimetry (DSC) was used to measure the melting (T-m), devitrification (T-d) and glass transition (T-g) temperatures of solutions with a range of compositions typical of those used for growing protein crystals, with the addition of glycerol as cryoprotectant. The addition of cryoprotectant raises the T-g and lowers the T-m of bulk solution thereby decreasing the cooling rates required for vitrification of protein crystals. The theoretical T-g value was calculated using the apparent volume fraction using the Miller/Fox equation extended for multi-component systems. The experimental values of T-g were within approximately +/- 4% of that predicted by the model. Thus, the use of the model holds the promise of a rational method for the theoretical determination of the composition of cryoprotectant requirement of protein crystallization solutions. (c) 2006 Elsevier B.V. All rights reserved.