The effect of tetrandrine on the electrically induced elevation of cytosolic Ca 2+ concentration, [Ca 2+] i, in the single isolated rat radiomyocyte was studied with a fluorometric ratio method using fura-2 acetomethylester (fura-2/AM) as Ca 2+ indicator. Tetrandrine (3–100 μM) concentration and time dependently inhibited the amplitude of the [Ca 2+] i transient without any significant effect on the resting level of [Ca 2+] i. At high concentration (60–100 μM), tetrandrine also prolonged the time to reach the peak ( t 1.0) and the time to decline to 20% of the peak level ( t 0.2) of the electrically induced [Ca 2+] i transient. The effect of tetrandrine was fast in onset and fully reversible upon washout. Tetrandrine (10 μM) partially inhibited the elevation of [Ca 2+] i in response to KCl-induced depolarization. Verapamil and diltiazem mimicked the effects of tetrandrine given at low concentrations, but not at high concentrations. At high concentrations, tetrandrine reduced the magnitude of the caffeine-induced [Ca 2+] i transient. Tetrandrine (100 μM) administered after thapsigargin, which itself decreased the amplitude and prolonged the duration of the electrically induced [Ca 2+] i transient, further decreased the amplitude of the [Ca 2+] i elevation. After ryanodine, which itself decreased the amplitude of the [Ca 2+] i transient, 100 μM tetrandrine not only further reduced the amplitude, but also prolonged the duration of the electrically induced [Ca 2+] i transient. These results provide evidence that in addition to its inhibitory effect on Ca 2+ influx at the sarcolemma at the therapeutically relevant concentrations, tetrandrine at high concentrations may inhibit Ca 2+ uptake into the sarcoplasmic reticulum.