Studies have suggested that integration of kinase and phosphatase activities maintains the steady-state L-type Ca²⁺ current in ventricular myocytes, a balance disrupted in failing hearts. As we have recently reported that the PP1/PP2A inhibitor, calyculin A, evokes pronounced increases in L-type I_Ca, the goal of this study was to identify the counteracting kinase and phosphatase that determine "basal" I_Ca in isolated mouse ventricular myocytes. Whole-cell voltage clamp studies, with filling solutions containing 10 mM EGTA , revealed that calyculin A (100 nM) increased I_Ca at test potentials between -42 and +49 mV (44% at 0 mV) from a holding potential of -80 mV. It also shifted the V_0.5 of both activation (-17 mV to -25 mV) and steady-state inactivation (-32 mV to -37 mV) in the hyperpolarizing direction. The broad-spectrum protein kinase inhibitor, staurosporine (300 nM), was without effect on I_Ca when added after calyculin A. However, by itself, staurosporine decreased I_Ca throughout the voltage range examined (50% at 0 mV) and blocked the response to calyculin A, indicating that the phosphatase inhibitor's effects depend upon an opposing kinase activity. The PKA inhibitors Rp-cAMPs (100 µM in the pipette) and H89 (1 µM) failed to reduce basal I_Ca or to block the calyculin A-evoked increase in I_Ca. Likewise, calyculin A was still active with 10 mM intracellular BAPTA or when Ba²⁺ was used as the charge carrier. These data eliminate roles for PKA and CaMKII as counteracting kinases. However, the PKC inhibitors Ro 31-8220 (1µM) and Gö 6976 (200 nM) decreased steady-state I_Ca and blunted the effect of calyculin A. PP2A is not involved in this regulation as intracellular applications of neither 10-100 nM okadaic acid nor 500 nM fostriecin increased ICa. However, PP1 is important as dialysis with 2 µM okadaic acid or 500 nM inhibitor-2 mimicked the increases in I_Ca seen with calyculin A. These in situ studies identify constitutive activity of PP1 and the counteracting activity of certain isoforms of PKC, in pathways distinct from receptor-mediated signalling cascades, as regulatory components that determine the steady-state level of cardiac L-type I_Ca.