The purpose of this study was to investigate the site of production (urothelium/smooth muscle) and quantitative release of prostanoids in the ureter. Quantitative analysis of prostacyclin (PGI2) was by its metabolite, 6-keto PGFalpha, thromboxane (TXB2) and prostaglandin F2alpha. Synthesis by radiometry and radioimmunoassay was performed in vitro in sheep ureter specimens before and after removal of the inner epithelial layer and after addition of indomethacin. The major prostanoids present in the ureter were PGI2 and TXB2; PGI2 was quantitatively the largest component. Removal of the thin inner epithelial layer (urothelium) reduced mostly PGI2; addition of arachidonic acid significantly augmented PGI2 only in ureters with intact urothelium but did not alter TXB2 levels. The main source of prostanoid synthesis (PGI2) of the ureter is to be found in the urothelium. The functional role of the prostanoids may be related to coordination of peristalsis.