Introduction: The macroscopic volume-regulated anion current (VRAC) is regulated by both intracellular and extracellular ATP, which has important implications in signaling and regulation of cellular excitability. The outwardly rectifying Cl^- channel (ORCC) is a major contributor to the VRAC. The purpose of this study was to investigate the effects of intracellular and extracellular ATP on the ORCCs expressed in the human cardiovascular system. Methods and Results: Using inside-out single channel patch clamp techniques, ORCCs were recorded from myocytes isolated from the human atrium, septal ventricle and from primary cells originating from human coronary artery endothelium and human coronary artery smooth muscle. ORCCs from all of these tissues had similar biophysical properties, i.e. they were outwardly rectifying, exhibited a slope conductance of 90~100 pS at positive potentials and ~22 pS at negative potentials, and they had a high open probability that was independent of voltage or time. The presence of ATP at the cytosolic face of the membrane increased the number of patches that contained functional ORCC, but had no effect on gating. In contrast, "extracellular" ATP (in the pipette solution) had no effect on the proportion of patches in which ORCC was detected, but strongly reduced gating. The potency order for nucleotides to affect gating was ATPγS>ATP=UTP>ADP>AMP, which suggests that a negatively charged phosphate group is involved in ORCC block. Conclusions: Our findings are consistent with a role of ORCC in the human cardiovasculature (atrium, ventricle and coronary arteries). The regulation of ORCC by extracellular ATP suggests that this channel may have an important role in maintaining electrical activity and membrane potential under conditions in which extracellular ATP levels are elevated, such as with ATP release from nerve endings or during pathophysiological conditions.