Using cultures of beating cardiomyocytes from neonatal rats, we have studied the fatty acid composition of phosphatidylinositol, phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate and the effect of supplementing the culture medium with docosahexaenoic acid on the fatty acid composition of the three phosphoinositides. Docosahexaenoic acid was incorporated into the phosphatidylinositol fraction of the supplemented cells, but not into the phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate fractions. At complete confluence, the cardiomyocytes were stimulated with an alpha 1-agonist (phenylephrine). This altered the acidic pattern of the phosphoinositides in both control and supplemented cells. The differences observed between the polyphosphorylated classes and the phosphatidylinositol fraction suggest the existence of different mechanisms of selection of fatty acids in the biosynthesis of phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate.