Isoproterenol (ISO), a beta agonist, causes hyperpolarization of coronary smooth muscle cells via an increase in K⁺ conductance. This hyperpolarization may cause the coronary vasorelaxation by decreasing the cytoplasmic Ca²⁺ concentration. It is well known that the activation of beta adrenoreceptors stimulates the adenylate cyclase activity, and the resulting K⁺ channel phosphorylation by cAMP-dependent protein kinase may be responsible for ISO-induced increase in K⁺ channel activity. However, it is not clear whether the increase in K⁺ channel activity by ISO is exclusively due to the activation of adenylate cyclase or not. In this research, the effect of ISO on the isometric tension and the mechanism of ISO-induced K⁺ channel activation were investigated in various patch clamp conditions. The summarized results are as follows. ISO- and pinacidil induced vasorelaxation was significantly inhibited by the application of TEA or by increasing the external K⁺ concentration. In the whole cell clamp mode, application of ISO increased K⁺ outward current, and this effect was completely eliminated by propranolol. In the cell-attached patch, application of ISO or forskolin increased Ca²⁺-activated K⁺ channel activity. Application of ISO to the bath in the outside-out patches or GTP in the inside-out patches stimulated Ca²⁺-activated K⁺ channels. From the above results, both A-kinase dependent channel phosphorylation and direct GTP-binding protein mediated effect might be responsible for the the activation of Ca²⁺-activated K⁺ channel by ISO in rabbit coronary smooth muscle cells. And this K⁺ channel activation also contributes to the ISO-induced vasorelaxation.