Single ventricular myocytes paced at a constant rate and held at a constant temperature exhibit beat-to-beat variations in action potential duration (APD). In this study we sought to quantify this variability, assess its mechanism, and determine its responsiveness to electrotonic interactions with another myocyte. Interbeat APD90 (90% repolarization) of single cells was normally distributed. We thus quantified APD90 variability as the coefficient of variability, CV = (SD/mean APD90) x 100. The mean ± SD of the CV in normal solution was 2.3 ± 0.9 (132 cells). Extracellular TTX (13 μM) and intracellular EGTA (14 mM) both significantly reduced the CV by 44 and 26%, respectively. When applied in combination the CV fell by 54%. In contrast, inhibition of the rapid delayed rectifier current with L-691,121 (100 nM) increased the CV by 300%. The CV was also significantly reduced by 35% when two normal myocytes were electrically connected with a junctional resistance (R(j)) of 100 MΩ. Electrical coupling (Rj = 100 MΩ) of a normal myocyte to one producing early after depolarization (EAD) completely blocked EAD formation. These results indicate that beat-to-beat APD variability is likely mediated by stochastic behavior of ion channels and that electrotonic interactions act to limit temporal dispersion of refractoriness, a major contributor to arrhythmogenesis.