Objective: Diacetyl monoxime (DAM), a nucleophilic agent with “phosphatase-like” activity, has been found to effectively and reversibly block cardiac muscle contraction, while the cells remain capable of generating transmembrane action potentials. The aim of this study was to characterise the effects of DAM on the electrical properties of cardiac muscle. Methods: Sheep epicardial muscle, guinea pig papillary muscle, and guinea pig ventricular myocytes were studied using conventional microelectrode techniques as well as single electrode current and voltage clamp techniques. Results: DAM (5-20 mM) decreased action potential duration at 50% and 90% repolarisation levels (APD50, APD90) and refractory period in a dose dependent manner without causing significant changes in action potential amplitude, maximum upstroke velocity, or resting membrane potential. DAM induced a slight decrease in action potential conduction velocity in both the longitudinal and transverse directions, but on average the conduction velocity recorded in the presence of the drug was not significantly different from control. The time course of the APD restitution curve was not significantly changed but the frequency dependent APD variations were reduced. The ionic bases for these changes were studied in guinea pig ventricular myocytes. As with the results obtained in tissue preparations, DAM 15 mM decreased APD50and APD90 by 35% and 29%, respectively. Under voltage clamp conditions, DAM led to a 35% reduction of ICa. The delayed rectifier IK current and the inward rectifier background current were also partially depressed by DAM but to a lesser extent. All of these effects were reversible upon washout. Conclusions: Aside from its well known effect as an electromechanical uncoupler, DAM causes a small, reversible, and non-selective reduction of several membrane conductances. Provided such effects are taken into consideration, DAM is a valuable tool in electrophysiological studies. Cardiovascular Research 1993;27:1991-1997