In many symptomatic patients, cholesterolosis of the gallbladder in the absence of gallstones can be responsible for the symptoms; however, it is difficult to establish its diagnosis despite repeated cholecystography or ultrasonography. The purpose of this prospective study was to evaluate the presence of cholesterol crystals in duodenal bile analysis for the diagnosis of cholesterolosis of the gallbladder. Two groups of subjects were analyzed: 16 asymptomatic subjects with normal cholecystography and 28 patients with suspected acalculous cholesterolosis (biliary symptoms and cholecystography and ultrasonography with minimal defects at the internal surface of the gallbladder wall). All controls showed absence of cholesterol crystals in duodenal bile, while 22 of the symptomatic patients had positive cholesterol crystals in duodenal bile. This finding was correlated with acalculous cholesterolosis in 90% of them. These patients demonstrated supersatured cholesterol content in bile compared to the control group.