The effects of isosorbide-5-mononitrate (IS-5-MN) and isosorbide-2-mononitrate (IS-2-MN) on vascular smooth muscle tone and on cyclic nucleotide levels were studied in isolated dog aorta. The effects of the mononitrates were compared with that of the relatively well-studied vasorelaxing agent sodium nitroprusside (NP). The substances induced concentration-dependent relaxation of the aortic rings contracted with noradrenaline. The potency order was: NP greater than IS-2-MN greater than IS-5-MN. IS-5-MN and IS-2-MN did not cause significant changes in the cAMP levels in dog aortic preparations. In sharp contrast, definite rises in the cGMP levels were observed in dog aortic rings after treatment with the mononitrates. With respect to both phenomena, IS-2-MN proved to have a stronger effect compared to IS-5-MN. These data support the hypothesis that an increase in cGMP is responsible for the relaxation of vascular smooth muscle by organic nitrates.