Here a comparison is made between adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl current (I_Cl) density and activation time course in response to -adrenoceptor stimulation with isoproterenol and adenylyl cyclase activation with forskolin. Saturating concentrations of isoproterenol and forskolin failed to activate an I_Cl in guinea pig atrial as well as in rat and frog ventricular cardiomyocytes. In guinea pig ventricular cardiomyocytes, step application of 1 µM isoproterenol induced an I_Cl of 0.89 ± 0.32 pA/pF (holding potential 40 mV, temperature 22 ± 1°C). I_Cl activation started after 3 ± 1 s, was complete within 44 ± 9 s, and was abolished after cell dialysis with the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate. Stimulation with increasing concentrations of forskolin (0.01-10 µM) increased I_Cl density and accelerated I_Cl activation. With 1 µM forskolin, I_Cl density was maximal (0.57 ± 0.30 pA/pF) but significantly smaller than that achieved with 1 µM isoproterenol. Although I_Cl density could not be further augmented by forskolin >1 µM, current activation (latency 28 ± 8 s, full activation after 112 ± 8 s with 1 µM forskolin) was further accelerated by 3 and 10 µM forskolin. However, I_Cl activation with 10 µM forskolin was still slower than that with 1 µM isoproterenol. A low isoproterenol concentration (1 nM), which did not activate I_Cl by itself, accelerated the 1 µM forskolin-induced activation of I_Cl by 35%; this speeding up was abolished after cell dialysis with guanosine 5'-O-(2-thiodiphosphate). I_Cl deactivation after the washout of 1 µM forskolin or 1 µM isoproterenol followed a similar time course. After stimulation with 10 µM forskolin or 1 µM forskolin + 1 µM isoproterenol, but not with 1 µM forskolin + 1 nM isoproterenol, the decay of I_Cl was significantly delayed. These results indicate that both cAMP-dependent and cAMP-independent G protein pathways contribute to the regulation of guinea pig ventricular I_Cl.