1. Ca2+ release from the sarcoplasmic reticulum (SR) was examined in enzymatically isolated single guinea-pig ventricular myocytes by monitoring [Ca2+]i with fura-2 during whole-cell recording of action potentials at room temperature (23-25 degrees C). Modulation of Ca2+ release by the Na+ current (INa) was studied by manipulating Na+ influx through the Na+ channel. 2. For a comparable Ca2+ loading of the SR, brief hyperpolarizing currents applied at the peak of the action potential increased Ca2+ release, while depolarizing pulses had the opposite effect. Similar currents applied before the action potential did not affect Ca2+ release. 3. Application of tetrodotoxin (TTX; 60 microM) moderately reduced Ca2+ release from the SR, but this effect was delayed in comparison with the immediate block of INa. An early effect of TTX was to increase Ca2+ release. 4. Replacement of Na+ with Li did not reduce Ca2+ release, but led to a progressive increase in Ca2+ release, resulting in spontaneous activity. 5. Ca2+ channel blockers (CdCl2, 100 microM; nisoldipine, 20 microM; or nifedipine, 20 microM) drastically reduced Ca2+ release from the SR. 6. Voltage clamp experiments confirmed that TTX blocked INa and its associated [Ca2+]i transient during voltage steps from -90 to -50 mV. INa and its associated [Ca2+]i transient were equally suppressed following replacement of Na+ with N-methyl-D-glucamine (NMDG+), but the [Ca2+]i transient was not suppressed following replacement of Na+ with Li+. 7. The INa-associated transient was sensitive to Ca2+ channel blockers. During steps from -50 to 0 mV, it appeared that the dihydropyridine antagonists often did not provide full block of the calcium current (ICa). 8. During current clamp stimulation at 1 Hz in the presence of TTX (60 microM), the Ca2+ content of the SR was decreased, due to the changes in action potential configuration and to changes in [Na+]i. 9. Our experiments indicate that the Ca2+ entry coupled to Na+ influx via the Na+ channel does not contribute substantially to the trigger for Ca2+ release from the SR during action potentials (23-25 degrees C). However, INa modulates Ca2+ release by affecting the Ca2+ load of the SR.