Stress responses have been implicated in pathological cardiovascular conditions. To set up heat-stressed model, we incubated isolated aortic rings at 42 ��C for 45 minutes. After 4 hours from the end of the heat treatment, we performed contractile experiment and western blot analysis. Since MLCK and ROK pathways are main pathways which are activated by KCl, we tested the hypothesis that those pathways are over stimulated by heat treatment. After heat treatment, contractile response to KCl was significantly enhanced in isolated rat aorta. KCl-induced enhanced contraction was proportionally relaxed by ROK inhibitor Y27632 and MLCK inhibitor ML7 in a concentration and time-dependent manner respectively. Phosphorylation of MLC20 and MYPT1Thr696 by KCl were increased after heat-shock treatment and MLC20 phosphorylation was abolished by Y27632 and ML7. The inhibition of KCl-induced augmented contraction by pretreatment with ML7 was remarkably attenuated in heat-treated aorta. In contrast pretreatment with Y27632 proportionally inhibited KCl-induced contraction in heat-treated aorta. The heat-augmented contraction was abolished by Ca++ channel blocker Nifedipine and the aorta did not contract at all in Ca++ free condition. These results indicate that heat stress augments ca++-dependent ROK pathway which results in MLC20 phosphorylation and contraction in response to KCl. Accordingly, ROK pathway could be a therapeutic target for stress-associated cardiovascular disease.