The mechanism by which adenosine 5'-triphosphate (ATP) shortens atrial action potential duration was studied in a canine model in vivo. Previous studies have indicated that the negative chronotropic and dromotropic actions of ATP in the canine heart are mediated by a vagal reflex and by adenosine. However, the mechanism of ATP's action on atrial action potential duration remains unknown. The effects of ATP on endocardial monophasic action potential were determined under baseline conditions (control) and after left cervical vagotomy followed by right vagus nerve afferent blockade with capsaicin (1% in olive oil), and subsequent bilateral cervical vagotomy plus propranolol (1.0 mg/kg, i.v.). In addition, the effects of ATP and adenosine were determined 48 h following the administration of pertussis toxin (PTX, 30 micrograms/kg, i.v.). PTX intoxication was verified by monitoring plasma levels of insulin during glucose tolerance tests. ATP (4 and 6 mumol/kg, rapid bolus into right atrium) markedly shortened right atrial action potential duration at 50% repolarization (APD50) from 101 +/- 8 to 22 +/- 6 and from 111 +/- 8 to 14 +/- 2 ms, respectively. Adenosine (equimolar doses given in an identical mode) had a smaller effect, i.e., APD50 of 106 +/- 8 and 109 +/- 6 was shortened to 77 +/- 12 and 76 +/- 12 ms, respectively. Left cervical vagotomy slightly reduced the effect of ATP but not that of adenosine. Blockade of right vagal C fiber afferent traffic using local application of capsaicin to the right cervical vagosympathetic trunk markedly attenuated the effect of ATP, but not that of adenosine. Autonomic blockade (i.e., bilateral cervical vagotomy and propranolol) markedly attenuated the effect of ATP, but not of adenosine; for example, the effect of ATP (6 mumol/kg) was reduced from 86 +/- 2% shortening of APD50 to 24 +/- 5% (p < 0.05), while that of adenosine was 32 +/- 8 and 20 +/- 4% (ns) before and after autonomic blockade, respectively. Treatment with PTX completely abolished the effect of both ATP and adenosine on atrial action potential duration. These data indicate that (i) the effect of ATP on the canine atrial action potential duration is mediated to a large extent by a vagal reflex triggered by the nucleotide and to a lesser extent by adenosine, the product of ATP's enzymatic degradation,(ii) the afferent traffic of this reflex travels mainly via the right vagal C fibers, and (iii) the effects of both vagal and the adenosine components are mediated by PTX-sensitive guanine nucleotide binding proteins (G proteins) coupled to the muscarinic cholinergic receptors and A1 adenosine receptors, respectively.