Gastrin Releasing Peptide (GRP) has a widespread distribution and multiple stimulating effects on endocrine and exocrine secretions and metabolism. The prohormone for GRP (ProGRP, 125 amino acids) is processed to the amidated, biologically active end products GRP_1-27 and GRP_18-27. Amidated forms of GRP are putative autocrine or paracrine growth factors in a number of cancers including colorectal cancer. However the potential role and biological activity of proGRP has not been investigated. Using a newly developed antisera directed to the N terminus of human proGRP, proGRP immunoreactivity was detected in all of the endometrial, prostate and colon cancer cell lines tested and in 9/10 resected colorectal carcinomas. However no amidated forms were detected suggesting an attenuation of processing in tumors. Recombinant proGRP was expressed as a His-tag fusion protein and purified by metal affinity chromatography and HPLC. ProGRP stimulated proliferation of a colon cancer cell line and activated mitogen-activated protein kinase but unlike GRP_18-27amide had no effect on inositol phosphate production. ProGRP did not compete with iodinated bombesin in binding assays on Balb 3T3 cells transfected with the known GRP receptors, GRP-R or BRS-3. We conclude that proGRP is present in a number of cancer cell lines and in resected colorectal tumors and is biologically active. Our results suggest that antagonists to GRP precursors rather than the amidated end products should be developed as a treatment for colorectal and other cancers that express proGRP-derived peptides.