ABSTRACT. The inotropic effect of diazepam, a benzodiazepine derivative, and its mechanism of action were examined using guinea pig heart and single ventricular cell preparations. In Langendorff hearts and right ventricular free-wall preparations, diazepam (10 to 100 µM) produced a monophasic negative inotropic effect in a concentration dependent manner. Neither a central type (flumazenil 1 µM) nor a peripheral type (PK11195 10 µM) of benzodiazepine receptor antagonist antagonized the monophasic negative inotropic effects of diazepam. Diazepam (10 to 100 µM) shortened action potential duration of papillary muscle in a concentration dependent manner. In isolated single ventricular cells, diazepam (30 and 100 µM) inhibited the calcium current (I Ca ) in a concentration dependent manner. Diazepam produced a significant decrease in I Ca elicited by first depolarizing pulses, however, the decrease of I Ca was not augmented during a train of depolarizing pulses. Thus, diazepam appears to produce a tonic block of cardiac calcium channels and the mode of inhibition is clearly different from the use-dependent block of verapamil. From these results, it was concluded that diazepam produces a monophasic negative inotropic effect that is independent of the benzodiazepine receptor, and is probably mediated through an inhibition of I Ca in guinea pig heart preparations.KEY WORDS: calcium current, diazepam, heart, negative inotropic effect, patch clamp method.