Objective: Contractions of isolated, single myocytes of guinea pig heart stimulated at 37 o C consist of a phasic component and a voltage dependent tonic component. In this study we investigated the source of Ca 2 + activating the tonic component. Methods: Experiments were performed at 37 o C in ventricular myocytes of guinea pig heart. Voltage-clamped cells were stimulated by the pulses from the holding potential of -40 to +5 mV. [Ca 2 + ] i was monitored as fluorescence of Indo 1-AM and contractions were recorded with the TV edge-tracking system. Results: Superfusion of 5 mmol/l Ni 2 + during 30 s pause did not inhibit subsequent biphasic Ca 2 + transients and contractions despite inhibition of Ca 2 + current and Na + /Ca 2 + exchange. KB-R7943 (5 μmol/l) or intracellular dialysis with 0 Na + solution, both of which inhibit reversed Na + /Ca 2 + exchange, decreased amplitude of Ca 2 + transients and contractions by ~40%. The ratio of amplitudes of tonic to phasic component was increased by Ni 2 + and was not changed by KB-R7943 or 0 Na + i . Ryanodine (200 μmol/l) inhibited both components of contractions in cells superfused with Ni 2 + . The phasic component but not the tonic component was inhibited by 20 μmol/l nifedipine in cells superfused with Ni 2 + . Conclusions: Tonic component of contraction of single myocytes of guinea pig heart is not activated by Ca 2 + current or by the reverse mode Na + /Ca 2 + exchange as currently proposed in literature. Rather, it is activated by Ca 2 + released from the sarcoplasmic reticulum. However, kinetics and mechanism of release seem to be quite different from those of Ca 2 + fraction activating the phasic component of contraction.