This study 1) compares the negative chronotropic and dromotropic actions of adenosine in guinea pig, rat, and rabbit hearts; 2) investigates the mechanism(s) for the different responses; and 3) determines the physiological implications. Isolated perfused hearts were instrumented for measurement of atrial rate and atrioventricular (AV) nodal conduction time. Differences in metabolism of adenosine were determined in the absence and presence of dipyridamole (nucleoside uptake blocker) and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, adenosine deaminase inhibitor). Dipyridamole plus EHNA decreased adenosine's EC50 for the negative dromotropic effect by 14-fold in guinea pig heart and 1.6-fold in rat heart. This is consistent with the greater number of [3H]nitrobenzylthioinosine binding sites measured in membranes from guinea pig (1,231 +/- 68 fmol/mg protein) compared with rat (302 +/- 31 fmol/mg protein) and rabbit (260 +/- 28 fmol/mg protein) atria. The potency of adenosine to slow atrial rate and prolong AV nodal conduction time was greater in guinea pig than in rat or rabbit hearts. This rank order of potency correlated well with the number of binding sites for the specific adenosine receptor radioligand 125I-aminobenzyladenosine in guinea pig (102 +/- 13 fmol/mg protein), rat (11 +/- 0.5 fmol/mg protein), and rabbit (8 +/- 1 fmol/mg protein) atrial membranes. Hypoxia increased the rate of adenosine release by severalfold and caused slowing of heart rate and AV block. In spontaneously beating hearts, the main effect of hypoxia was a slowing of ventricular rate, which in the guinea pig heart was due to AV block and in the rat heart to atrial slowing. In atrial paced hearts, hypoxia caused a marked prolongation of AV nodal conduction time in guinea pig (39 +/- 4 msec) and rabbit (29 +/- 5 msec) hearts, but only small effect in rat hearts (10 +/- 2 msec). The differences in response to hypoxia could be accounted for by the species-dependent differences in the 1) amount of adenosine released and metabolized, 2) sensitivity of the hearts to adenosine, and 3) dependency of AV nodal conduction on atrial rate. The findings indicate that the results from physiological or pharmacological studies on adenosine in one species may not be applicable to others, and the ultimate effect of adenosine and hypoxia is to slow ventricular rate.