The bioactivity of stearidonic acid (SDA, 18:4n-3) and alpha-linolenic acid (LNA, 18:3n-3) on cyclooxygenase-2 (COX-2) enzyme expression and prostaglandin E2 (PGE2) production has not been evaluated. This investigation examined the effects of SDA and LNA on PGE2 biosynthesis and COX-2 protein and mRNA levels in MDA-MB-231 human breast cancer cells. Cells were supplemented with SDA, LNA, linoleic acid and arachidonic acid (AA) at concentrations ranging from 10 to 200 microM. At 50 and 200 microM, both SDA and LNA treatments and their combinations reduced PGE2 production as compared with AA. At 50 microM, SDA treatment also lowered the COX-2 protein level as compared with the vehicle, but this reduction was not observed with the LNA treatment. Gas chromatographic analysis of fatty acids in cellular lipids of breast cancer cells revealed that SDA led to significantly greater concentrations of 20:5n-3 and other long-chain (LC) n-3 polyunsaturated fatty acids (PUFAs) (20:4n-3, 22:4n-3 and 22:5n-3) as compared with the LNA treatment. Both SDA and LNA reduced the level of 20:4n-6; however, SDA was more effective than LNA in decreasing the ratio of n-6/n-3 PUFAs in cells. In addition, SDA was more potent than LNA in suppressing the expression of the COX-2 gene, which was associated with the reduction in the levels of nuclear factor kappa B and peroxisome proliferator-activated receptor gamma mRNA. This study showed that although PGE2 production in MDA-MB-231 breast cancer cells was not significantly different between the SDA and LNA treatments, SDA was more effective than LNA in converting into LC n-3 PUFAs and in reducing COX-2 protein and mRNA levels.