Polyunsaturated fatty acids are involved at several steps in the turnover of phosphatidylinositol mediated by alpha 1-adrenoceptors. We have cultured neonatal rat cardiomyocytes in a medium containing docosahexaenoic acid (C22:6n3) and have investigated the effects produced by the change in the fatty acid composition of phosphatidylinositol on alpha 1-adrenoceptor-mediated phosphatidylinositol metabolism. The experimental cells, in which phosphatidylinositol was significantly enriched in docosahexaenoic acid and depleted in arachidonic acid, showed a reduced ability to incorporate labeled inositol into phospholipids in comparison with control cells, both in basal conditions and after stimulation by phenylephrine. This decreased incorporation led to a reduced availability of inositol phospholipids, substrates of phospholipase C, and to a reduced production of inositol phosphates under basal conditions.