Mechanisms for the antiarrhythmic effect of n-3 polyunsaturated fatty acids (PUFA) are currently being investigated using isolated cardiac myocytes. It is still not known whether the incorporation of n-3 PUFA into membrane phospholipids is a prerequisite for its protective action or if n-3 PUFA exert antiarrhythmic effects in their nonesterified form as demonstrated by recent studies. Adult porcine cardiomyocytes were grown in media supplemented with arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). After 24 h, analysis of total lipids showed that the myocytes were enriched with the respective fatty acids compared to control cells. Large proportions of all three fatty acids supplemented (69% AA, 72% DHA, and 66% EPA) remained unesterified. Fatty acid analyses of total phospholipids (PL) revealed that the incorporation of EPA and DHA, though small, was significantly different (P<0.05) from that of the control cells. The PL fraction was further separated into phosphatidylinositol (PI), phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine to study the pattern of incorporation of the fatty acids in these fractions. It became apparent that EPA and DHA were selectively incorporated into the PI fraction. This study demonstrates that in adult porcine cardiomyocytes, the n-3 PUFA supplementation selectively modulates two important lipid fractions, nonesterified fatty acid and PI, which were implicated in the mechanisms of prevention of cardiac arrhythmias.