Effects of intracellularly perfused ATP, and extracellularly applied cyanide and 2-deoxy-D-glucose, on fast and slow Ca2+channel currents of isolated single vascular smooth muscle cells were investigated by a whole-cell voltage-clamp method combined with an intracellular perfusion technique. Single smooth muscle cells were prepared by collagenase treatment from guinea pig small mesenteric arteries (diameter of <300 μm). With Cs+-rich solution in the pipette and isotonic Ba2+solution (100 mM) in the bath, depolarizing pulses evoked two types of the Ca2+channel current. Depolarizing pulses from the holding potential of -80 mV to over -30 mV evoked a fast Ca2+channel current. This fast component was inhibited by shifting the holding potential in a positive direction. With a holding potential of -40 mV, the fast component was almost inhibited. In contrast, the slow current was evoked by command potentials to above -10 mV, and its full amplitude was preserved at the holding potential of -40 mV. Without ATP in the pipette, the fast current was dominant. Increase in the ATP concentration in the pipette (0.3 to 5 mM) enhanced the slow current but did not affect the fast current. Maximum enhancement of the slow current was observed at 5 mM ATP. Increase in ATP concentration, however, did not modify the shape of the current trace and the steady state inactivation curve of the slow current. Maximum amplitudes of the fast current and slow current recorded with 5 mM ATP averaged 17.4 pA (SD of 10.4 pA, n=30; observed at -10 mV to +10 mV) and 141.8 pA (SD of 27.1 pA, n=30; observed at +30 mV to +40 mV), respectively. Presence of CN˜ and 2-deoxy-D-glucose (without glucose) in the bath, and absence of ATP in the pipette, abolished the slow current within 10 minutes; in contrast, it took more than 10 minutes to depress the fast current. The inhibitory effect of CN-and 2-deoxy-D-glucose on the slow current was reduced by intracellular application of ATP. In summary, the activation of the slow Ca2+channel required physiological concentration of ATP, whereas the fast channel current was preserved, even under ATP-free conditions. These results indicate that only the slow current is a metabolically dependent Ca2+channel current in these vascular smooth muscle cells.