The eicosanoid profiles, sites of production, and response to bradykinin stimulation were determined in rabbit ileum and its various components by radioimmunoassay of various prostanoids and 5-lipoxygenase products in the incubation media. The profile of eicosanoid synthesis and secretion by the epithelial cell fraction was PGF2 alpha greater than 6-keto-PGF1 alpha greater than dihydro-keto-PCM = PGE2 greater than TxB2 much greater than 5-HETE greater than LTB4 and by the deepitheliated ileum was PGE2 = 6-keto-PGF1 alpha greater than PGF2 alpha greater than dihydro-keto-PGM greater than TxB2 much greater than LTB4 greater than 5-HETE. PGD2 was not sought in these studies. Rates of eicosanoid production by the deepitheliated ileum were over 200 times that of the epithelial cells. The epithelial cells accounted for 67% of the protein but only 0.2% of the PGE2 produced, while the lamina propria and submucosa contained only 12-30% of the protein but produced 80-90% of the PGE2. Bradykinin (1 microM), A23187 (10 microM), arachidonic acid (20 microM), and melittin (0.7 microM) stimulated PGE2 and 6-keto-PGF1 alpha production by 200% in deepitheliated (or subepithelial) ileum, but bradykinin failed to stimulate production of any eicosanoid by the epithelial cell fraction. Thus the subepithelium (predominantly the lamina propria) is the major eicosanoid producer of rabbit ileum and is the major site of bradykinin-stimulated eicosanoid synthesis and secretion. Eicosanoids released from subepithelial components may be important regulators of epithelial function.