Measurements were made of trans-sarcolemmal Ca(2+) fluxes and intracellular [Ca(2+)](i) in rat ventricular myocytes loaded with Indo-1 to determine how the n-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) suppresses spontaneous waves of Ca(2+) release. We report that in 10 microM EPA, the Ca(2+) efflux generated by individual waves increased by 11.3 +/- 4.9 % over control levels. However, wave-generated efflux per unit time fell overall by 19 +/- 5.3 %. On removal of EPA, wave frequency increased transiently such that Ca(2+) efflux was greater than normal and the cell lost 28.0 +/- 10.6 micromol l(-1) Ca(2+). This probably represents the loss of extra Ca(2+) accumulated by the sarcoplasmic reticulum (SR), while Ca(2+) release was inhibited. These results are evidence of inhibition of the SR Ca(2+)-release mechanism and reduced availability of Ca(2+) to the SR. From the relationship between average intracellular Ca(2+) and the frequency of spontaneous waves, we have calculated the relative contributions of these different mechanisms to the lower frequency of waves. In EPA, the frequency of spontaneous waves fell by 37.5 +/- 8.1 %, the majority of this (29.2 +/- 8.8 %) is due to inhibition of the Ca(2+)-release mechanism. In EPA, the rate of fall of Ca(2+) in the caffeine response (an indicator of surface membrane Ca(2+) efflux pathway activity) was not altered. We conclude, therefore, that the lower resting level of Ca(2+) observed in EPA is due to a lower influx of Ca(2+) across the surface membrane rather than increased activation of efflux pathways. How these effects might contribute to the anti-arrhythmic actions of EPA is discussed.