Heat shock at temperatures from 38°C to 44°C for 2 or 4 h conferred two varieties of maize seedlings an acquired thermotolerance. Pretreatment of maize seeds with CaCl2 solution significantly enhanced this heat-shock induced thermotolerance, but MgCl2 treatment had little effect. On the contrary, Ca2+ chelator EGTA treatment lowered this heat-shock induced thermotolerance. In addition, treatment with Ca2+ channel blockers La3+ or verapamil also weakened the heat-shock induced and Ca2+-enhanced thermotolerance in the maize seedlings. On the other hand, treatment with calmodulin antagonists CPZ or W7 greatly eliminated the heat-shock induced and Ca2+-enhanced thermotolerance, while W5, a less active analogue of W7, had little effect. In addition, calmodulin level in the maize coleoptiles increased remarkably during heat shock. Ca2+ level in the seedlings affected their calmodulin level and thermotolerance. These results suggest that Ca2+ and calmodulin may be involved in the acquisition of the heat-shock induced thermotolerance. The acquisition of heat-shock induced thermotolerance requires the entry of extracellular Ca2+ into cells across the plasma membrane and the mediation of intracellular calmodulin.