The mechanism of extracellular adenosine 5'-triophsphate (ATP)-triggered vagal depressor reflex was further studied in a closed chest canine model. Adenosine and ATP were administered individually in equimolar doses (0.01-1.0 :mol/kg) into the right (RCA) and left circumflex (LCA) coronary arteries. When given into the RCA, adenosine and ATP exerted an identical and relatively small negative chronotropic effect on sinus node automaticity; the time-to-peak-effect of which was 7 sec. When given into the LCA, adenosine was without effect while ATP markedly suppressed sinus node automaticity. This effect of ATP, (i) reached its peak in less than two seconds following its administration, (ii) was short lasting and (iii) was completely abolished by either intravenous administration of the muscarinic cholinergic blocker atropine (0.2 mg/kg) or intra-LCA administration of 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP), a potent P2X_2/3 purinergic receptor (P2X_2/3R) antagonist, but not by diinosine pentaphosphate (Ip₅I), a potent inhibitor of P2X₁R and P2X₃R. Repetitive administrations of ATP were not associated with reduced effects indicative of receptor desensitization, thereby excluding the involvement of the rapidly desensitized P2X₁R in the action of ATP. It was concluded that ATP triggers a cardio-cardiac vagal depressor reflex by activating P2X_2/3R located on vagal sensory nerve terminals localized in the left ventricle. Since these terminals mediate vasovagal syncope, these data could suggest a mechanistic role of extracellular ATP in this syndrome, and in addition, give further support to the hypothesis that endogenous ATP released from ischemic myocytes is a mediator of atropine sensitive bradyarrhythmias associated with left ventricular myocardial infarction.