Previous studies demonstrated that the naturally occurring electrophile and PPARgamma ligand, nitrolinoleic acid (NO(2)-LA), exists as a mixture of four regioisomers [Alexander, R. L., et al. (2006) Biochemistry 45, 7889-7896]. We hypothesized that these alternative isomers have distinct bioactivities; therefore, to determine if the regioisomers are quantitatively or qualitatively different with respect to PPARgamma activation, NO(2)-LA was separated into three fractions which were identified by NMR (13-NO(2)-LA, 12-NO(2)-LA, and a mixture of 9- and 10-NO(2)-LA) and characterized for PPARgamma interactions. A competition radioligand binding assay showed that all three NO(2)-LA fractions had similar binding affinities for PPARgamma (IC(50) = 0.41-0.60 microM) that were comparable to that of the pharmaceutical ligand, rosiglitazone (IC(50) = 0.25 microM). However, when PPARgamma-dependent transcription activation was examined, there were significant differences observed among the NO(2)-LA fractions. Each isomer behaved as a partial agonist in this reporter gene assay; however, the 12-NO(2) derivative was the most potent with respect to maximum activation of PPARgamma and an EC(50) of 0.045 microM (compare with the rosiglitazone EC(50) of 0.067 microM), while the 13-NO(2) and 9- and 10-NO(2) derivatives were considerably less effective with EC(50) values of 0.41-0.62 microM. We conclude that the regioisomers of NO(2)-LA are not functionally equivalent. The 12-NO(2) derivative appears to be the most potent in PPARgamma-dependent transcription activation, whereas the weaker PPARgamma agonists, 13-NO(2) and 9- and 10-NO(2), may be relatively more important in signaling via other, PPARgamma-independent pathways in which this family of nitrolipid electrophiles is implicated.