A collection of evidence suggests that conjugation of double bonds of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, omega-3 polyunsaturated fatty acids (n-3 PUFAs), increases their anticarcinogenic activity; however, the effect of such conjugation on vascular tone activity remains unknown. We propose that the mixture of conjugated PUFAs exerts higher vasorelaxation activity than the corresponding mixture of nonconjugated PUFAs. The vascular response to different concentrations of conjugated and nonconjugated isomers of EPA and DHA, among other fatty acids (FAs) naturally present in shark oil, and the role of nitric oxide (NO) as a vasorelaxant agent were investigated. Both conjugated EPA (CEPA) and conjugated DHA (CDHA) were prepared by alkaline isomerization of all PUFAs contained in shark oil. Different concentrations of conjugated and nonconjugated PUFAs were placed in contact with precontracted aortic rings of Wistar rats to assess their effect on vascular tone. All tested samples exerted a vasorelaxant effect. Compared to nonconjugated PUFAs, conjugated isomers exhibited an increase in the dilatation of the aortic rings (P<0.001) in a dose-dependent manner (P<0.001). In addition, nonconjugated PUFAs produced nitric oxide (NO) in a dose-dependent manner, while conjugated PUFAs did not, suggesting that their dilatation mechanism is not totally dependent on NO.