The present study was undertaken to demonstrate and characterize potentiation of ventricular overdrive suppression by adenosine. To substantiate that adenosine has an enhanced effect on overdrive suppression, it would be necessary to demonstrate that adenosine increases pause duration independent of slowing spontaneous pre-drive rate. In isolated perfused guinea pig hearts with surgically induced complete atrioventricular block, the effect of adenosine (2-20 microM) on pause duration was compared to two alternative means of slowing the pre-drive rate, i.e., hypothermia (28.0 degrees C to 34.0 degrees C) and cesium chloride (0.3-1.0 mM). The slope value of the linear regression line describing the relationship between pre-drive cycle length and pause duration for adenosine (15.8) was significantly greater than control (1.7), hypothermia (1.7), and cesium chloride (5.4). The competitive adenosine antagonist, aminophylline (60 microM), when infused at the initiation of overdrive during adenosine administration, significantly reduced the effect of adenosine on pause duration by 72.9 +/- 4.2% (mean +/- SEM). The reduction in pause duration by aminophylline was specific for adenosine and did not occur under control conditions or during cesium chloride administration. During hypoxia, aminophylline and adenosine deaminase, when infused at the initiation of overdrive, caused 72.3 +/- 5.6 and 63.3 +/- 6.1% reductions in pause duration, respectively. Endogenous adenosine levels rose significantly with hypoxia (1,687 +/- 202 vs. 36 +/- 4 pmol/min per g during normoxia) and increased significantly further during hypoxic overdrive (3,004 +/- 323 pmol/min per g). In isolated guinea pig Purkinje fibers (n = 4), adenosine (20 microM) increased pause duration by 73.6 +/- 9.9% while only minimally affecting the pre-drive cycle length (7.6 +/- 3.8%). These fibers, when stimulated at 1.5 Hz, also displayed an adenosine-induced reduction in action potential duration at 90% repolarization (16 +/- 2 msec). In addition, we demonstrated that adenosine had an enhanced effect on pause duration in the presence of ouabain (1 microM)-induced attenuation of overdrive suppression. Thus, in isolated Purkinje fibers, it is unlikely that the potentiating effect of adenosine on pause duration, which is independent of its chronotropic effect, is mediated via an enhancement of sodium potassium adenosine triphosphatase pump activity. The effect of adenosine is likely to be secondary to a direct action on outward potassium conductance.(ABSTRACT TRUNCATED AT 400 WORDS)