This study examines the role of nitric oxide (NO) in the regulation of calcium absorption in the small intestine. Calcium absorption was quantified by measuring 45Ca++ transport from lumen to blood in an intestinal segment (duodenum and 20 cm of the proximal jejunum) perfused by both intraluminal and vascular routes in anesthetized rats. When administered i.v. as bolus injections, NG-nitro-L-arginine methyl ester (L-NAME, 10 mg.kg-1), an inhibitor of NO biosynthesis, decreased calcium absorption with a concomitant increase in blood pressure and a decrease in mesenteric blood flow. Conversely, the nitrovasodilators 3-morpholinosydnonimine (2 mg.kg-1) and S-nitroso-N-acetylpenicillamine (10 micrograms.kg-1), which generate NO spontaneously, both increased calcium absorption with no change in mesenteric blood flow. When infused i.v., L-NAME (3 mg.hr-1.kg-1 for 40 min) induced a decrease in calcium absorption that was reversed by the NO donor sodium nitroprusside (1.5 mg.hr-1.kg-1 when infused for the last 20 min of the 40-min L-NAME infusion). Sodium nitroprusside infusion (1.5 mg.hr-1.kg-1) caused an increase in calcium absorption that was not reversed by L-NAME (3 and 30 mg.hr-1.kg-1). The present findings suggest that NO is involved in basal calcium absorption in rat small intestine in vivo.