Omega-6 (n-6) arachidonic acid (AA) and its pro-inflammatory metabolites, including prostaglandin E2 (PGE2), are known to promote tumorigenesis. Delta-6 desaturase (D6D) is the rate-limiting enzyme for converting n-6 linoleic acid (LA) to AA. Our objective was to determine if AA synthesis, specifically D6D activity, and PGE2 levels are increased in cancerous breast tissue, and whether these variables differ between estrogen receptor positive (ER+) and negative (ER−) breast cancers. Gas chromatography was performed on surgical breast tissue samples collected from 69 women with breast cancer. Fifty-four had ER+ breast cancer, and 15 had ER− breast cancer. Liquid chromatography-mass spectrometry was used to determine PGE2 levels. Lipid analysis revealed higher levels of LA metabolites (C18:3 n-6, C20:3 n-6, and AA) in cancerous tissue than in adjacent noncancerous tissue (P < 0.01). The ratio of LA metabolites to LA, a measure of D6D activity, was increased in cancerous tissue, suggesting greater conversion of LA to AA (P < 0.001), and was higher in ER− than in ER+ patients, indicating genotype-related trends. Similarly, PGE2 levels were increased in cancerous tissue, particularly in ER− patients. The results showed that the endogenous AA synthetic pathway, D6D activity, and PGE2 levels are increased in breast tumors, particularly those of the ER− genotype. These findings suggest that the AA synthetic pathway and the D6D enzyme in particular may be involved in the pathogenesis of breast cancer. The development of drugs and nutritional interventions to alter this pathway may provide new strategies for breast cancer prevention and treatment.