A critical step in the formation of cholesterol gallstones is nucleation (i.e., the formation of cholesterol monohydrate crystals from supersaturated bile). The rate of nucleation of cholesterol depends upon a critical balance between pronucleating and antinucleating factors in bile. Mucin, a high molecular weight glycoprotein secreted by the gallbladder and biliary duct epithelium, is a pronucleating agent in experimental and human gallstone disease. Gallbladder mucin shares with other epithelial mucins the ability to bind lipids and bile pigment. The hydrophobic binding sites in the polypeptide core of mucin may provide a favorable environment for nucleation of cholesterol monohydrate from supersaturated bile. In nearly all animal models of cholelithiasis, mucin hypersecretion is prominent. The stimulus for gallbladder mucin hypersecretion appears to be a component of lithogenic bile. Prostaglandins regulate mucin release in gallbladder epithelium in vitro and probably in vivo. In the cholesterol-fed prairie dog, blockage of mucin release with aspirin inhibits gallstone formation. These findings suggest that inhibition of mucin release may prevent cholesterol stone formation during high-risk periods or after dissolution therapy with bile salts.