1. Propranolol, as the racemate and the (+)- and (-)-isomers (400 mug/l.) and practolol (50 mg/l.) were tested for their effects on atrial and ventricular rates and on the duration of overdrive suppression (ODI) in isolated perfused cat hearts with surgically-induced heart block.2. Racemic propranolol and the (+)- and (-)-isomers prolonged ODI and slowed the rate of the ventricular pacemaker; the (+)- and (-)-isomers also reduced the rate of the atrial pacemaker. Practolol shortened ODI and increased the rate of the atrial and ventricular pacemakers.3. The (+)- and (-)-isomers were more potent than the racemate; the (-)-isomer was more potent than the (+)-isomer. The results suggest there is a stereospecific mechanism involved in the biological distribution of propranolol.4. Twenty-four hours after reserpine treatment (5 mg/kg, i.p.) practolol continued to increase the rate of the atrial and ventricular pacemakers but did not shorten ODI.5. The mechanism by which these agents affect myocardial excitability and automaticity is discussed.