We examined and compared mechanisms of the red wine (RW)-induced vasorelaxation in guinea pig (GP) and rat aorta. Acetylcholine-induced relaxation of norepinephrine-precontracted aortic rings was stronger in rat aorta than in GP aorta, whereas RW-induced vasorelaxation was stronger in GP aorta. l-nitro-arginine methyl ester (l-NAME) abolished RW-induced vasorelaxation in rat aorta, whereas in GP aorta, it was only reduced by 50%. To examine mechanisms of the l-NAME-resistant relaxation, GP aortic rings were additionally exposed to indomethacin, clotrimazole, and their combination. Indomethacin insignificantly reduced RW-induced relaxation, but in combination with l-NAME, the relaxation was synergistically decreased (80%). After clotrimazole exposure, the relaxation was reduced by 25%, and addition of indomethacin caused no further reduction. Only the combination of l-NAME, indomethacin, and clotrimazole prevented RW-induced vasorelaxation. RW-induced vasorelaxation in KCl-precontracted GP rings was significantly smaller (Emax 78.31% ± 6.09%) than the RW-induced relaxation in norepinephrine-precontracted rings (Emax 126.01% ± 2.11%). l-NAME in KCl-precontracted GP rings prevented RW-induced vasorelaxation. In conclusion, different pathways are involved in the RW-induced vasorelaxation in GP aorta, in contrast to rat aorta, in which NO plays main role. Therefore, the uncritical extrapolation of the results from one species to another could be misleading.