Nonmucous glycoproteins as pronucleating agents. Cholesterol crystallization-promoting factors probably play an important role in the pathogenesis of gallstone disease. We have isolated one of the factors involved by using lectin-affinity chromatography. A potent promoting activity binds to concanavalin A-Sepharose. The activity is heat labile and sensitive to digestion by glycosidase but remarkably insensitive to proteases. The concanavalin A-binding pronucleator affects cholesterol solubilization in model bile in two ways. It induces a shift of cholesterol and phospholipid from the micellar to the vesicular phase but also interacts directly with cholesterol-phospholipid vesicles. The concanavalin A-binding protein fraction contains at least two different promoting factors with gel permeation molecular weights of about 150 kD and 5 kD, respectively. The higher molecular weight activity could be assigned to a protein with an apparent molecular weight of 130 kD. Concanavalin A-binding-promoting activity was present in bile from both patients with and without stones, indicating that it is a normal constituent of bile. However, the activity was strongly increased in bile from patients with multiple cholesterol gallstones, suggesting that it could play a key role in gallstone formation in these patients. 