A recent report by DuBell & Rogers, (2004) showed that while inhibition of protein phosphatases 1 and 2A (PP1/PP2A) with calyculin A increased basal I_Ca, 1μM H-89 (an inhibitor of PKA) had no effect, leading to the conclusion that PKA-mediated phosphorylation was not responsible for sustaining I_Ca observed in the absence of catecholamines. However, earlier studies have demonstrated that 10μM H-89 (Yuan & Bers, 1995) decreased the forskolin activated I_Ca in ferret ventricular myocytes. We therefore investigated the effects of H-89 in more detail to determine the role of constitutive PKA in regulating basal I_Ca. Myocytes were isolated from the ventricles of humanely killed rats using collagenase and protease digestion and voltage clamped with Axopatch-200B amplifier using amphotericin B (240μg ml^-1 ) in the perforated patch clamp configuration. Myocytes were preloaded with 20μM Bapta AM and stimulated by 400 ms square pulses from −40 mV to 60 mV in 5 mV increments at 0.5 Hz. Internal pipette solutions contained (mM): 120 Cs glutamate, 20 KCl, 10 NaCl, 1.0 CaCl₂, 10 Hepes, pH 7.2. The external Tyrodes solution contained 140 NaCl, 5.4 KCl, 1.0 MgCl₂, 1.0 CaCl₂, 10 Hepes, pH 7.3. Recordings were performed at 35-37^oC. Inhibition of PP1/2A by external application of 1μM calyculin A increased peak I_Ca by 51% from −1.23 ±0.16 nA to −1.86 ± 0.17 nA (n=11; P<0.05), whereas isoprenaline (also 1 μM) increased I_Ca by 181% (n=5, P<0.05). Inhibition of PKA using H-89 produced a concentration-dependent decrease in peak I_Ca with an EC₅₀ of 8 mM (n=8, P<0.05). I_Ca recorded during double pulse protocols and fitted with a Boltzmann equation, to determine activation (d) and inactivation (f) variables, showed that in the presence of 10μM H-89, f was shifted from −15.1± 0.6 to −18.2 ± 1.1 mV (n=8), whereas d remained unchanged (−6.5 ±1.4 mV in control and −8.6 ± 1.1 mV, n=8). An additional series of double-pulse protocols examined the effects of H-89 on the time-dependent recovery state of I_Ca from voltage-dependent inactivation. Under control (n=10) conditions 50% recovery (R₅₀) of I_Ca took 515.4 ±62.2 ms, whereas in the presence of H-89 (10μM; n=8) R₅₀ significantly (P<0.05) increased by 93% to 994.0 ± 129.2 ms. Data show that in rat ventricular myocytes, H-89 significantly decreased basal I_Ca amplitude, shifted voltage-dependent inactivation to more negative values and slowed time-dependent recovery from voltage-dependent inactivation. Constitutive PKA-mediated phosphorylation is therefore most likely to be responsible for the basal I_Ca amplitude and activation/inactivation kinetics even in the absence of β-adrenergic receptor stimulation.