We have purified a protein from serum-free conditioned medium of the HT29 human colon adenocarcinoma cell line based on its ability to inhibit the proliferation of the same cell line. The purification procedure consisted of acid gel permeation, semipreparative, and analytical reversed-phase chromatographies. The high-pressure liquid chromatography-purified colon cancer cell growth inhibitor migrates as a single band of 27 and 34 kDa on sodium dodecyl sulfate/polyacrylamide gels under nonreducing and reducing conditions, respectively. NH2-terminal amino acid sequence analysis of the first 32 residues has demonstrated that this protein belongs to the insulin-like growth factor-binding protein (IGFBP) family. More precisely, this growth inhibitor appeared to be identical to the recently cloned human IGFBP-4. This IGFBP (HT29-IGFBP) has been characterized by performing ligand blotting and competitive binding experiments. The affinity of HT29-IGFBP for insulin-like growth factor (IGF) II (approximately 3.4 x 10(10) M-1) is slightly greater than its affinity for IGF-I (approximately 1.4 x 10(10) M-1). HT29 cells also produce two other isoforms (28 and 31 kDa, nonreduced) of the HT29-IGFBP having the same partial NH2-terminal amino acid sequence as the 27-kDa protein. The monoclonal antibody alpha IR-3 is known to block the mitogenic actions of IGFs. alpha IR-3 inhibited the growth of HT29 cells, thus suggesting that IGFs are required for the growth of these colon cancer cells.