Gallbladder biles and stones were obtained at 116 cholecystectomies for symptomatic gallstone disease. All 33 patients younger than 50 years had cholesterol stones, whereas 40% of the older patients had pigment stones. We compared the reliability of three different bile tests for the differentiation between cholesterol and pigment stone patients. Whereas both the presence of cholesterol monohydrate crystals in fresh gallbladder bile and a nucleation time ⩽20 days in ultrafiltered gallbladder bile had a specificity of 100% for cholesterol gallstone disease, biliary supersaturation with cholesterol (cholesterol saturation index >1.0) had a low specificity. The sensitivity of nucleation time ⩽20 days for cholesterol gallstone disease was 78% in concentrated gallbladder biles (biliary total lipid concentration ⩾5 g/dl) but only 21% in dilute biles (biliary total lipid concentration <5 g/dl). In contrast, examination for the presence of cholesterol crystals in fresh bile was reasonably sensitive both in concentrated and diute gallbladder biles (sensitivity, 84% and 72%, respectively). In addition, duodenal bile obtained from 16 patients (10 cholesterol, 6 pigment) before cholecystectom showed cholesterol crystals in 7 of the cholesterol but in none of the pigment stone patients. We conclude that examination of fresh bile for cholesterol crystals is a specific and reasonably sensitive test for cholsterol gallstone disease.