Amidated gastrin-releasing peptide (GRP) is the prototypical autocrine growth factor. Nonamidated peptides derived from the C terminus of pro-GRP are also biologically active in colorectal cancer (CRC) cell lines in vitro, via a receptor distinct from the GRP receptor. The aims of this study were to measure the amounts of pro-GRP-derived peptides in human CRC cell lines and tumors, characterize the immunoreactive peptide, and investigate its effect on proliferation in vitro and in vivo. Pro-GRP-derived peptides were quantitated by region-specific ELISA in extracts of five human CRC cell lines and 20 tumors. The immunoreactive material was purified by HPLC and its mass and sequence established by mass spectrometry. The concentration of GRPamide was determined by RIA. Proliferation of DLD-1 cells and murine gastrointestinal mucosa was measured by [3H]-thymidine incorporation and mitotic index, respectively. In CRC cell extracts, ELISA for pro-GRP-derived peptides detected 3–152 fmol/106 cells. The immunoreactive peptide was purified and identified as pro-GRP42–98. Resected stage III tumors contained significantly less pro-GRP immunoreactivity than stage II tumors, and no amidated GRP was detected in cell lines or tumors. Stable transfection of DLD-1 cells with pro-GRP short hairpin RNA, or treatment with a monoclonal anti-pro-GRP antibody, significantly reduced proliferation. Pro-GRP42–98, pro-GRP47–68, and pro-GRP80–97 significantly stimulated mitosis in colonic, but not small intestinal, mucosa of 10-wk-old mice. We conclude that nonamidated peptides derived from the C terminus of pro-GRP are expressed in significant quantities in CRC cell lines and tumors and stimulate the proliferation of CRC cells and of normal colonic mucosa. Such peptides are attractive targets for novel CRC therapies.