The effects of glycerol and water content on the thermal transitions of plasticized barley starch were examined using differential scanning calorimetry. The glycerol contents studied were 14, 20, 29 and 39% and the water content, obtained by conditioning in different relative humidities, varied in the range 1–28%. On the basis of the observed calorimetric glass transition temperatures and corresponding heat capacity increments it was inferred that a single phase system occurred at low water and glycerol contents, while in other cases phase separation occurred and the system was composed of starch-rich and starch-poor phases. Dynamic mechanical thermal analysis on a phase-separated sample showed mechanical loss peaks corresponding to the glass transitions of both phases. Amylopectin crystallization did not occur within 1 week of storage in mixtures having less than 20% water, indicating that glycerol interacted with starch, inhibiting crystallization of amylopectin.