OBJECTIVE Peroxidation of membrane lipids, altering cell integrity and function, plays an important part in the onset and development of cardiac damage following ischemia and reperfusion. Cells maintain their membrane lipid homeostasis by substituting peroxidized lipids with new polyunsaturated fatty acids. The microsomal enzymatic system converting essential fatty acids to highly unsaturated fatty acids (HUFAs) contributes to this repairing mechanism. The membrane of the endoplasmic reticulum could be one of the potential targets of free radicals generated in ischemia/reperfusion, thus causing a reduced efficacy of the system required for HUFA biosynthesis. To verify this hypothesis, and the consequent modification in fatty acid composition, we exposed cultured rat cardiomyocytes to different periods of hypoxia (H), eventually followed by reoxygenation (R). Furthermore, the effectiveness of antioxidants like alpha-tocopherol and a green tea extract in counteracting H/R damage towards HUFA biosynthesis was tested. RESULTS Linoleic (LA) and alpha-linolenic acid (ALA) conversion was measured by pre-labelling cells with [1-14C]LA or [1-14C]ALA for 1 h; total lipid fatty acid composition was determined by gas chromatographic analysis. H profoundly affected HUFA biosynthesis, and this effect was much more evident on LA than on ALA. Conversion of both substrates was partially restored during R due to the readmission of the final acceptor of the desaturating complex. Fatty acid composition data were in agreement with the modifications observed in essential fatty acid conversion. Antioxidant protection appeared to be related to the duration of H, and to be more effective during H than during R. CONCLUSIONS This study points out the importance of possessing good antioxidant defenses not only after, but mainly prior to the onset of H.