The human colon contains many open-type endocrine cells which express the preproglucagon gene and possess glucagon-like peptide-1 (GLP-1) immunoreactivity, but the molecular form of the peptide is unknown. Acid ethanol extracts of human colon (n = 4) were subjected to gel filtration and successive purification by high-pressure liquid chromatography, monitored by specific RIAs. A single GLP-1-immunoreactive peak was isolated and identified as GLP-1 (7-36)amide by amino acid sequence analysis and mass spectrometry. We conclude that proglucagon is processed in the large intestine in the same manner as in the small intestine, and results in the formation of fully processed biologically active GLP-1.