Human colonic mucin has been isolated from normal colonic mucosa by a phenol-water extraction procedure and purified by Sepharose 2B column chromatography. The mucin was further purified by cesium bromide density gradient centrifugation. Sodium dodecyl sulfate-polyacrylamide gel (5%) electrophoresis of this material showed high-molecular-weight mucin component(s) at the top of the gel. Chemical analyses of this preparation indicated a typical mucin profile of amino acids and carbohydrates. Ion-exchange chromatography resulted in the separation of two major fractions, one being more acidic than the other. Chemical deglycosylation of the purified preparation at 20 degrees C for 3 1/2 showed loss of sialic acid, fucose, galactose, and N-acetylglucosamine, whereas traces of N-acetylgalactosamine were still detected. High-pressure liquid chromatography of the deglycosylated material resulted in the purification of a major peptide, P1, with high levels of threonine, serine, and proline, resembling, in most respects, the profile of native mucin. The molecular weight of the peptide was determined to be approximately 97 kDa and serine was the single NH2 terminus.