Blocking of l-type Ca channels by highly hydrophilic dihydropyridines, NKY-722 and KV-1360, was investigated in single ventricular cells of guinea-pig hearts using the whole-cell voltage clamp technique. At a holding potential of −30 mV, NKY-722 (1–100 n m) decreased the amplitude of the l-type Ca channel current (I Ca) in a concentration-dependent manner. NKY-722 did not change the time constants of the decay of I Ca. In the presence of NKY-722 (1 μ m), the steady-state inactivation curve was shifted toward a more negative potential (by −33.0±2.0 mV) without changing its slope factor. The use-dependent block was elicited at a pulse frequency of 3.3 Hz or more. Even after washing out the drug at −80 mV for 20 min. I Ca inhibited by NKY-722 (100 n m) at −30 mV was scarcely recovered when the membrane potential was clamped back to −30 mV. A permanently charged compound KV-1360 (0.1–1 μ m), a quaternary amine derivative of NKY-722, hardly affected I Ca by intracellular and extracellular application. These results suggest that, in spite of the high degree of ionization (91% in the charged form at pH 7.4), the mode of the l-type Ca channel block by NKY-722 is quite similar to that by lipophilic dihydropyridines. Consequently, the neutral form of NKY-722 is the active compound and this reaches the dihydropyridine receptor by “membranous approach”.