Background & Aims: Processing of progastrin, the 80-amino acid precursor of the hormone gastrin, generates a variety of peptides with distinct distributions and biological activities. However, little is known regarding the expression, secretion, and biological activity of the 6-amino acid C-terminal flanking peptide (CTFP) of progastrin. The objectives were to determine the concentration of CTFP in normal subjects and patients with gastrointestinal diseases and to investigate the biological activity of CTFP. Methods: CTFP, gastrin-amide (Gamide), glycine-extended gastrin (Ggly), and progastrin were measured using region-specific radioimmunoassay (RIA) in antral extracts and resected colorectal cancers (CRC) and in plasma from normal subjects (fasting and meal stimulated) and from patients with CRC, multiple endocrine neoplasia type 1 (MEN-1), or pernicious anemia. The effect of CTFP on proliferation, migration, and activation of the mitogen-activated protein kinase (MAPK) pathway in several types of gastrointestinal cell lines was determined. Results: CTFP is by far the predominant progastrin-derived peptide found in the antrum (4-fold higher than Gamide), resected CRC, and circulation (60-fold higher than Gamide) and is released after meal stimulation. The hypergastrinemic patients (MEN-1, pernicious anemia) had elevated plasma Gamide but unaltered CTFP demonstrating differential secretion of these 2 progastrin-derived peptides. Finally, CTFP stimulated proliferation and migration and activated MAPK of cells in culture. Conclusions: The high and regulated expression of CTFP in healthy and diseased subjects combined with the evidence for biological activity of CTFP demonstrates that CTFP is not an inactive metabolite of progastrin processing but is a bioactive peptide with potential roles in the normal and diseased gastrointestinal tract.