The fatty acids regulate gene expression directly binding to nuclear receptors or affecting the protein content of transcription factors. In this work, supplementing primary cultures of neonatal rat cardiomyocytes with 60 microM EPA or DHA, we demonstrated by an ELISA assay an increased PPAR beta/delta binding to DNA. n-3 PUFA supplementation deeply changed the acyl composition of both cytosolic and nuclear fractions. The high content of total fatty acids, particularly EPA and DHA, and its increase following supplementation suggested a selective accumulation of n-3 PUFAs in the nucleus, supporting the direct interaction of n-3 PUFA with PPAR. The activity of acyl-CoA thioesterase (ACOT), catalyzing the reaction leading to NEFA from acyl-CoA, increased in n-3 PUFA supplemented cells. The NEFA/acyl-CoA ratio is an important regulator of the fatty acid transport to the nucleus and consequent modulation of gene transcription, and although ACOT activity is not the only parameter of this ratio, it is important for the control of the NEFA pool composition. Our data further clarify what happens in cardiomyocytes following n-3 PUFA supplementation, linking the modification of acyl composition to ACOT activity and PPAR activation.