Radiolucent gallstones are common in young adults with cystic fibrosis. In the mid-1970s, it was suggested that gallstones are made of cholesterol, but this hypothesis has never been tested. Several recent studies have shown that the detection of cholesterol monohydrate crystals in bile has high sensitivity and specificity for the diagnosis of cholesterol gallstones. We therefore used this approach to study 17 young adults with cystic fibrosis, 10 of whom had radiolucent gallstones. The two groups of patients were comparable in age and gender (all patients but one were male). Duodenal bile was obtained after gallbladder contraction with intravenous cerulein; it was used for lipid and protein chemistry studies and for polarizing microscopy. The latter was performed both in whole bile and in the postultracentrifugation (100,000 g) sediment. Bile cholesterol saturation did not significantly differ between patients with (1.21 +/- 0.28) or without gallstones (0.99 +/- 0.54). Slight cholesterol supersaturation was found in 7 of 10 gallstone and three of seven nongallstone patients. At no time were cholesterol crystals detected in either the group, even after bile ultracentrifugation. Two more cystic fibrosis patients with gallstones died of severe bronchopneumopathy, and small pigment gallstones were obtained at autopsy. At stone analysis, cholesterol content was 44% and 28% of dry weight, respectively. Infrared spectroscopy of stone powder was compatible with the presence of calcium bilirubinate and proteins as major components. We conclude that radiolucent gallstones of cystic fibrosis are not of the conventional cholesterol type.