Isolated rat thoracic aortic strips undergoing noradrenaline-induced contraction were treated with an adult heartworm (HW) crude extract and then examined for isometric changes in tension. HW extract caused relaxation of endothelium-intact strips, but not endothelium-denuded strips. This effect was inhibited by treatment with NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) and could be reversed by additional treatment with L-arginine. However, HW extract at a high concentration caused slight relaxation of endothelium-denuded strips, and relaxation persisted after L-NAME treatment in endothelium intact-strips. These data suggested that the relaxation induced by HW extract was mainly endothelium-dependent, nitric oxide-mediated, but in part, also endothelium-independent. In addition, a bioassay using isolated rat thoracic aortas may be a useful tool for investigating vasoactive substances in the HW extract.