Myocardial ischemia-reperfusion activates the Na⁺/H⁺ exchanger, which induces arrhythmias, cell damage, and eventually cell death. Inhibition of the exchanger reduces cell damage and lowers the incidence of arrhythmias after ischemia-reperfusion. The -3 polyunsaturated fatty acids (PUFAs) are also known to be cardioprotective and antiarrhythmic during ischemia-reperfusion challenge. Some of the action of PUFAs may occur via inhibition of the Na⁺/H⁺ exchanger. The purpose of our study was to determine the capacity for selected PUFAs to alter cardiac sarcolemmal (SL) Na⁺/H⁺ exchange. Cardiac membranes highly enriched in SL vesicles were exposed to 10-100 µM eicosapentanoic acid (EPA) or docosahexanoic acid (DHA). H⁺-dependent ²²Na⁺ uptake was inhibited by 30-50% after treatment with 50 µM EPA or 25 µM DHA. This was a specific effect of these PUFAs, because 50 µM linoleic acid or linolenic acid had no significant effect on Na⁺/H⁺ exchange. The SL vesicles did not exhibit an increase in passive Na⁺ efflux after PUFA treatment. In conclusion, EPA and DHA can potently inhibit cardiac SL Na⁺/H⁺ exchange at physiologically relevant concentrations. This may explain, in part, their known cardioprotective effects and antiarrhythmic actions during ischemia-reperfusion.