To investigate the electrophysiological effects of propranolol in vivo over a wide range of plasma concentrations and to distinguish effects due to beta-blockade from those due to a direct membrane action, His bundle electrograms were recorded, and ventricular effective refractory periods (VERP) and monophasic action potential duration (MAP) were measured in anesthetized control dogs and in dogs given three graded infusions of d- or dl-propranolol. Dogs were excluded if the plasma concentrations attained did not fall in predefined ranges of 25--125, 125--700, and 700--3,000 ng/ml. Isoproterenol sensitivity tests were performed to determine the relative beta-blocking potency of the isomers at the three concentration ranges. The highest concentration of d-propranolol had approximately the same beta-blocking potency as the lowest concentration of dl-propranolol. Mean AH and HV intervals in the His bundle electrogram increased with the concentration of dl-propranolol, and the increase was greater than with d-propranolol (p less than 0.03) at the first and second concentration steps but not significantly different at the highest concentrations of d- and dl-propranolol. VERP and MAP increased directly with concentration of d- and dl-propranolol. Although the mean increases of VERP and MAP tended to be greater in the dl-propranolol group, the differences between d- and dl-propranolol were not statistically significant at any concentration. We conclude that prolongations of atrioventricular and His-Purkinje conduction are stereospecific responses and are due to beta-blockade. The specific mechanism for the prolongation of ventricular repolarization and refractoriness are effects of propranolol that could not be definitively classified in this study.