Single myocytes were obtained from guinea-pig ventricles, and the catecholamine-induced Cl- current (ICl) was recorded by the patch-clamp method combined with the use of caged adenosine 3',5'-cyclic monophosphate (cAMP) and the concentration jump technique. The rapid bath application of isoprenaline was followed by a marked latency of approximately 1.7 s before a sigmoidal onset of the ICl activation, while the photolysis of caged cAMP was followed by no measurable latency. The ICl conductance was increased by increasing the concentration of caged cAMP with a threshold of approximately 5 microM and a one-half effective concentration of 25 microM. On the other hand, the rate of the activation increased in proportion to the logarithm of the concentration of caged cAMP. When the ultraviolet flash was repeated or the myocytes were pretreated with either forskolin, 3-isobutyl-1-methylxanthine, or okadaic acid, the rate of the ICl activation accelerated, and the whole time course of the ICl activation became monoexponential. These findings suggest that both the production of cAMP and reactions on the multiple phosphorylation sites of the channel protein are major rate-limiting steps in the ICl activation.