Several endogenous peptides for G-protein-coupled receptors have been found to play physiological roles in muscle contraction in addition to their well-demonstrated actions in other tissues. To further identify such peptides, we screened over 400 peptides using an isometric tension assay of rat papillary muscle. Here, we report that kyotorphin, which is known as an analgesic dipeptide, has a cardiac effect. Although kyotorphin had no effect on the twitch tension itself, it inhibited beta-adrenergic agonist isoprenaline-induced increases in twitch tension in a dose-dependent manner. Leu-Arg, a selective antagonist of kyotorphin, reversed this inhibitory effect. The inhibitory effect was also reversed by naloxone, an opioid receptor antagonist. These results suggest that kyotorphin may release opioid peptides from rat cardiac muscle and have an indirect regulatory role in beta-adrenergic action through cross-talk with opioid receptors.