Goblet cell mucin (GCM) of rat small intestine has been isolated previously, and its location established by immunofluorescence techniques. In the present study, GCM was characterized more fully by chemical and physical methods. It was found to be a flexible negatively charged macromolecule with a molecular weight of about 2.0 × 106, an intrinsic viscosity of 15.3 dl/g, and an axial ratio of about 225:1. Its composition was protein 12%, total hexose 23%, hexosamine 22.4%, sialic acid 10%, fucose 6.6%, and sulfate < 1% of the dry weight. It contained approximately 34 disulfide bonds per molecule. Like most mucin glycoproteins it was rich in serine, threonine, and proline (45.5 mol %) and poor in hydrophobic and sulfur containing amino acids. One major and two minor components were identified by acrylamide disc gel electrophoresis and analytic ultracentrifugation. The components appeared to represent different molecular weight species of GCM. No evidence of subunit structure could be obtained using a variety of techniques, including the disruption of ionic, hydrophobic, and disulfide bonds by detergents, denaturants, or reducing agents.