Several mechanisms have been proposed to explain the anti-arrhythmic effects of n-3 polyunsaturated fatty acids. One mechanism is the effect of modifying cell membrane phospholipid and their subsequent effect on intracellular cell signaling via the second messengers, Ins(1,4,5)P(3) and diacylglycerol. Isolated cardiac myocytes from adult pig hearts were used to investigate the effect of n-3 polyunsaturated fatty acids, eicosapentaenoic acid and docosahexaenoic acid, on the inositol phosphate metabolism and protein kinase C activity. Adult porcine cardiac myocytes were grown in media supplemented with 400 µM arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid. After 24 hr, fatty acid analyses of total lipids by TLC in supplemented cells showed that eicosapentaenoic acid and docosahexaenoic acid were selectively incorporated into the phosphatidylinositol fraction. In the diacylglycerol fraction, there was a small incorporation of both eicosapentaenoic acid and docosahexaenoic acid but it was not significantly different from that of controls. To study the effect of membrane phospholipid modification on the phospholipase C mediated inositol lipid cycle, cardiac myocytes were labeled with 4µCi/ml myo-[2-(3)H]Ins for 48 hr. After stimulation with epinephrine and phenylephrine (alpha-receptor agonist) the water soluble [(3)H]Ins products were separated by chromatography on Dowex AG 1-X8 and measured by scintillation counting. After stimulation, the levels of [(3)H]Ins(1,4,5)P(3) and [(3)H]Ins(1,3,4,5)P(4) in eicosapentaenoic acid and docosahexaenoic acid supplemented myocytes were significantly reduced (P < 0.05) compared to arachidonic acid supplemented myocytes. Similarly, eicosapentaenoic acid and docosahexaenoic acid supplemented cells had reduced levels of protein kinase C activity after stimulation compared to arachidonic acid supplemented cells. From these experiments, it is evident that n-3 PUFA supplementation modulates intracellular cell signaling suggesting a possible anti-arrhythmic mechanism.