Involvement of ATP in the regulation of slow (L-type) Ca2+ channels of vascular smooth muscle cells was investigated by recording single Ca2+ channel currents (single-channel conductance of 18 pS) using a patch clamp technique. In the cell-attached configuration, intracellular composition was modified by permeabilizing the cell membrane with mechanical disruption at one end of the cell. Single cells were freshly isolated from guinea-pig portal vein by collagenase treatment. For the channel recordings, the pipette solution contained 100 mM Ba2+ and the bath contained K+-rich solution (with 5 mM EGTA) to depolarize the membrane to near 0 mV. The channel activity decreased usually within 3 min after permeabilizing the cell end and exposure to ATP-free bath solution. If ATP (1–5 mM) was applied to the bath (access to cell interior) before complete disappearance of channel activity, channel activity was partially recovered. ATP did not change the current amplitude (i) or the mean open time of the channels, whereas the number of channels available for opening and/or the probability of their being open (NP o) were increased by ATP. A non-hydrolyzable analogue of ATP, AMP-PNP, did not exert an ATP-like effect; ATP-γ-S had a weak effect. With 1 μM Bay-K-8644 (Ca2+ channel agonist) in the pipette, the activity of the Ca2+ channel was high; such activity persisted for more than 10 min after permeabilizing the cell and exposting to ATP-free solution containing KCN (1 mM) and 2-deoxy-d-glucose (10 mM). These results indicate that activation of slow Ca2+ channels requires ATP. The effect of ATP may be exerted by phosphorylation and/or an energy-requiring step. Bay-K-8644 may change the nature of the slow Ca2+ channel, making it resistant to rundown.