The origin of rat urinary epidermal growth factor (EGF) has been investigated. Unilateral nephrectomy decreased the concentration, total output of EGF and EGF/creatinine ratio by approximately 50%, while the output of creatinine was unchanged. Removal of the submandibular glands and duodenal Brunner's glands, organs known to produce EGF, had no influence on the output of EGF in urine. Renal clearance of EGF exceeded that of creatinine, and after bilateral nephrectomy or bilateral ligation of the ureters, the concentration of creatinine in serum increased, while the concentration of EGF was below the detection limit of the assay. Renal production of EGF was confirmed by immunohistochemistry demonstrating EGF immunoreactivity in the afferent arteriole of the juxtaglomerular apparatus. EGF in the submandibular glands and in urine was found to differ with chromatofocusing and reverse-phase HPLC. At isoelectric focusing the pI of submandibular EGF was 4.8 and 5.4 while that of urinary EGF was 5.3 and 6.4. In conclusion, this study demonstrates that urinary EGF mainly originates from the kidneys and is localized to the renal juxtaglomerular apparatus.