The purpose of this work was to study whether rat lactating mammary gland can synthesize PUFAs through the expression of Delta5 and Delta6 desaturases (Delta5D and Delta6D), whether these enzymes are regulated by the transcription factors sterol-regulatory element binding protein 1 (SREBP-1) and peroxisome proliferator-activated receptor alpha (PPARalpha) and the coactivator peroxisome proliferator-activated receptor gamma coactivator 1beta (PGC-1beta), and whether these desaturases are regulated by the lipid concentration in the diet. The results showed that on day 12 of lactation, approximately 35% of the linoleic acid in the diet, which is the precursor of PUFAs, is transferred to the mammary gland. There was expression of Delta5D and Delta6D in mammary gland, and it was regulated by the corn oil content in the diet. The higher the corn oil content in the diet, the lower the expression of both desaturases. Induction of Delta5D and Delta6D was associated positively with similar changes in SREBP-1 and PGC-1beta. Expression of PPARalpha was barely detected and was not affected by the corn oil content in the diet, whereas PGC-1beta expression increased as the corn oil in the diet increased. These results indicate that the lactating mammary gland has the capacity to synthesize PUFAs and can be regulated by the lipid content in the diet.