Aims: The present study is to investigate the mechanism by which ATP regulates Cav1.2 channel activity. Methods: Ventricular tissue was obtained from adult guinea pig hearts using collagenase. Ca²⁺ channel activity was monitored using the patch clamp technique. Proteins were purified using wheat germ agglutinin-Sepharose and the concentration was determined using the Coomassie Brilliant Blue technique. ATP binding to Cav1.2 channel was examined using the photoaffinity method. Results: EDA-ATP-Biotin maintains Ca²⁺ channel activity in inside-out membrane patches. ATP directly bound to Cav1.2 channel in a dose-dependent manner, and at least two molecules of ATP bound to one molecule of Cav1.2 channel. Low levels of CaM increased ATP binding to Cav1.2 channel, but higher levels of CaM decreased ATP binding to Cav1.2 channel. In addition, Ca²⁺ was another regulator for ATP binding to Cav1.2 channel. Furthermore, ATP bound to GST-fusion peptides of N-terminal region (amino acids 6-140) and proximal C-terminal region (amino acids 1509-1789) of the main subunit (α1C) of Cav1.2 channel. Conclusions: Our data suggest that ATP might regulate Cav1.2 channel activity by directly binding to Cav1.2 channel in a dose-dependent manner. In addition, ATP binding effect to Cav1.2 channel was both CaM- and Ca²⁺-dependent.