1. The effects of haemoglobin and methylene blue on the acetylcholine (ACh)-induced electrical and mechanical responses of smooth muscle cells were investigated in rat aorta and rat main pulmonary artery. 2. When the endothelium was intact, ACh induced a transient hyperpolarization and sustained relaxation of tissues precontracted with noradrenaline. Both hyperpolarization and relaxation were absent in preparations without endothelium. 3. Haemoglobin and methylene blue inhibited the ACh-induced relaxation, but not the transient hyperpolarization. 4. In aorta with an intact endothelium, ACh produced an increase in both the rate of 86Rb efflux and tissue cyclic GMP levels. The changes in ion flux were unaffected by either haemoglobin or methylene blue in concentrations which almost abolished the increase in cyclic GMP concentrations. 5. In arteries with an intact endothelium, indomethacin had no effect on the ACh-induced electrical and mechanical responses or on the increase in 86Rb efflux and tissue cyclic GMP levels. 6. It is concluded that in the rat aorta and rat main pulmonary artery, ACh releases two different substances, an endothelium-derived relaxing factor (EDRF) and a hyperpolarizing factor (EDHF), from the endothelial cells. Neither substance appears to be derived from a pathway dependent on cyclo-oxygenase. EDHF seems to play a minor role in the relaxation of noradrenaline-induced contractions.