Breast-fed mammals consume epidermal growth factor (EGF) in milk. We characterized processing of mouse EGF (mEGF) in gastrointestinal (GI) tract of 14-day-old auckling (Su) anesthetized mice using reversed phase-HPLC. 125I-mEGF (4-10 ng/mouse) in 50 μl of 0.05 M phosphate buffer + 0.1% BSA, pH 7.4 (intestine) or pH 5.5 (stomach) was introduced into ligated stomach, duodenum, jejunum, midjejunum or ileum. After 10 or 30 min, radioactivity in luminal flushings (LF) and wall of GI segments was extracted and analyzed by RP-HPLC. Standards ( 125I-mEGF-1: 1 amino acid less at C-terminus; 125I-mEGF-5: 5 amino acids less at C-terminus; and 125I-mEGF-6: 6 amino acids less at C-terminus) were prepared by controlled proteolysis (Endocrinology 118:875). Formation by the tissues of C-terminally processed 125I-mEGF was defined by coelution with standards. Isolated peaks were characterized by binding to anti EGF antibody and EGF-speciflc receptors. 125I-mEGF was found to be intact in LF of stomach; in LF of duodenum it was partially degraded to 125I-mEGF-1. In LF and wall of jejunum, midjejunum and ileum, 125I-mEGF-1, 125I-mEGF-5 and 125I-mEGF-6 were detected. All peaks isolated from GI tissues were fully immunoreactive. Receptor binding 125I-mEGF-I was significantly greater than that of intact 125I-mECF, whereas receptor bindings of 125I-mEGF-5 and 125I-mEGF-6 were significantly diminished. Thus luminally administered EGF undergoes C-terminal processing in the GI tract; type of biologically active fragments produced varies with segment.