Regional variation in the response of the thoracic aorta to contractile agonists has previously been demonstrated. Since the net contractile response reflects the interaction between smooth muscle activation and the release of endothelial substances, we hypothesize that agonist-stimulated release of endothelium-derived nitric oxide (NO) also varies along the length of the thoracic aorta. The distribution of thoracic aorta estrogen receptors is also regionalized. Since pregnancy augments the release of endothelium-derived NO by acetylcholine (ACh) in some arterial beds, we further hypothesize that pregnancy enhances the stimulated release of NO from the thoracic aorta. Aortae were removed from nonpregnant and near term pregnant guinea pigs and cut into ring segments numbered sequentially proximal to distal. The rings were suspended at their optimal passive tension and submaximally contracted with prostaglandin F<sub>2</sub><sub>α</sub>. Endothelium-derived NO-dependent relaxation to ACh increased moving proximal to distal along the aorta independent of pregnancy and ACh relaxation was unaffected by pretreatment with physostigmine to inhibit cholinesterase. The magnitude of the relaxation to carbachol among the different segments was similar to ACh. Pregnancy decreased the ED<sub>50</sub> for ACh of segments from the middle and distal segments of the thoracic aorta. Relaxation to the NO donor sodium nitropmsside and the nonreceptor-mediated endothelium-dependent relaxing agent A23187 was uniform along the length of the aorta and independent of pregnancy. These experiments demonstrate regional variation in the stimulated release of endothelium-derived NO in the guinea pig thoracic aorta which is increased by pregnancy.