The co-release of ATP with norepinephrine from sympathetic nerve terminals in the heart may augment adrenergic stimulation of cardiac Ca²⁺ channel activity. To test for a possible direct effect of extracellular ATP on L-type Ca²⁺ channels, single channels were reconstituted from porcine sarcolemma into planar lipid bilayers so that intracellular signaling pathways could be controlled. Extracellular ATP (2-100 µM) increased the open probability of the reconstituted channels, with a maximal increase of ~2.6-fold and an EC₅₀ of 3.9 µM. The increase in open probability was due to an increase in channel availability and a decrease in channel inactivation rate. Other nucleotides displayed a rank order of effectiveness of ATP > ,-methylene-ATP > 2-methylthio-ATP > UTP > adenosine 5'-O-(3-thiotriphosphate) >> ADP; adenosine had no effect. Several antagonists of P2 receptors had no impact on the ATP-dependent increase in open probability, indicating that receptor activation was not required. These results suggest that extracellular ATP and other nucleotides can stimulate the activity of cardiac L-type Ca²⁺ channels via a direct interaction with the channels.