Cinobufagin (CBG), a major bioactive ingredient of the bufanolide steroid compounds of Chan Su, has been widely used to treat coronary heart disease. At present, the effect of CBG on the L-type Ca(2+) current (I Ca-L) of ventricular myocytes remains undefined. The aim of the present study was to characterize the effect of CBG on intracellular Ca(2+) ([Ca(2+)]i) handling and cell contractility in rat ventricular myocytes. CBG was investigated by determining its influence on I Ca-L, Ca(2+) transient, and contractility in rat ventricular myocytes using the whole-cell patch-clamp technique and video-based edge-detection and dual-excitation fluorescence photomultiplier systems. The dose of CBG (10(-8) M) decreased the maximal inhibition of CBG by 47.93%. CBG reduced I Ca-L in a concentration-dependent manner with an IC50 of 4 × 10(-10) M, upshifted the current-voltage curve of I Ca-L, and shifted the activation and inactivation curves of I Ca-L leftward. Moreover, CBG diminished the amplitude of the cell shortening and Ca(2+) transients with a decrease in the time to peak (Tp) and the time to 50% of the baseline (Tr). CBG inhibited L-type Ca(2+) channels, and reduced [Ca(2+)]i and contractility in adult rat ventricular myocytes. These findings contribute to the understanding of the cardioprotective efficacy of CBG.