Essential fatty acids can be helpful in the prevention of several pathologies. The bioavailability of acute supplementation of different doses of flaxseed oil was studied by analyzing the level of alpha-linolenic acid (ALA) and linoleic acid (LA) in serum and tissues (adipose, liver) of rats tested at 2, 4, 8 and 16 h after the administration. The amount of flaxseed oil administered at increasing doses corresponded to 1, 2.5 and 5 g ALA/kg of body weight. The corresponding fatty acid methyl esters obtained via direct methylation were quantified using gas chromatography/mass spectrometry. Serum ALA level increased after 1 or 2.5 g/kg. ALA was increased in both adipose and liver tissue 4 h after the administration of 1 g/kg of flaxseed oil. There was no further increase by using a higher oil dosage. LA did not change in serum at the doses used.