Our recent data show that in cardiac myo-cytes polyunsaturated fatty acids (PUFAs) are antiarrhyth-mic. They reduce I Na , shorten the action potential, shift the threshold for excitation to more positive potentials, and prolong the relative refractory period. In this study we use patch-clamp techniques in whole-cell mode and confocal Ca 2؉ imaging to examine the effects of PUFAs on the voltage-gated L-type Ca 2؉ current (I Ca,L), elementary sarcoplasmic reticu-lum Ca 2؉-release events (Ca 2؉-sparks), and [Ca 2؉ ] i tran-sients in isolated rat ventricular myocytes. Extracellular application of eicosapentaenoic acid (EPA; C20:5 n ؊ 3) produced a prompt and reversible concentration-dependent suppression of I Ca,L. The concentration of EPA to produce 50% inhibition of I Ca was 0.8 M in neonatal rat heart cells and 2.1 M in adult ventricular myocytes. While the EPA induced suppression of I Ca,L , it did not significantly alter the shape of the current-voltage relation but did produce a small, but significant, negative shift of the steady-state inactivation curve. The inhibition of I Ca,L was voltage-and time-dependent, but not use-or frequency-dependent. Other PUFAs, such as docosahexaenoic acid, arachidonic acid, linolenic acid, linoleic acid, conjugated linoleic acid, and eicosatetraynoic acid had similar effects on I Ca,L as EPA. All-trans-retinoic acid, which had been shown to suppress induced arrhythmogenic activity in rat heart cells, also produced a significant inhibition of I Ca,L. The saturated stearic acid and the monounsaturated oleic acid had no effect on I Ca,L. Because both I Ca,L and sarcoplasmic reticulum Ca 2؉-release underlie many cardiac arrhythmias, we examined the effects of EPA on I Ca,L and Ca 2؉-sparks. While EPA suppressed both, it did not change the temporal or spatial character of the Ca 2؉-sparks, nor did it alter the ability of I Ca,L to trigger Ca 2؉-sparks. We conclude that PUFAs may act as antiarrhythmic agents in vivo in normal and Ca 2؉-overloaded cells principally because they reduce Ca 2؉ entry by blocking I Ca,L. Furthermore, PUFAs act directly to decrease I Na and I Ca,L , but indirectly to reduce the [Ca 2؉ ] i transients and [Ca 2؉ ] i-activated membrane current. Although a negative inotropic action is associated with application of PUFAs, it is clear that by reducing I Ca,L , I Na and Ca 2؉-sparks, PUFAs can reduce spontaneous extrasystoles in the heart. The mechanisms by which PUFAs act are discussed.