The purpose of this study was to determine whether mucous glycoprotein is the nucleating factor responsible for the rapid in vitro nucleation time of gallbladder bile from persons with cholesterol gallstones. Ultracentrifugation and ultrafiltration of abnormal bile removed all detectable mucous glycoprotein, yet bile that had been filtered exhibited as rapid a nucleation time as unaltered bile. When abnormal bile was heated to 95 °C for 60 min, nucleation time was significantly prolonged. Rapid nucleation time could be restored to heated abnormal bile by addition of small volumes of unheated bile. Purified human mucous glycoprotein accelerated nucleation time of human bile, but mucous glycoprotein from control patients was as effective as that from gallstone patients. There was a direct relationship between mucous glycoprotein concentration and effect on nucleation time. Mucous glycoprotein may be important in the early stages of stone formation, but it is probably not the agent responsible for the sharp discrimination between control bile and gallbladder bile from patients with cholesterol stones found in the in vitro nucleation time test. The markedly prolonged nucleation time of heated abnormal bile is preliminary evidence that the nucleating factor may be a heat-labile protein other than mucous glycoprotein.