The aggregative forms of lipids in human gallbladder bile and their relation to cholesterol crystallization are controversial. Using combined chemical, gel-chromatographic, optical/electron microscopic and quasielastic light-scattering methods, we investigated this issue in native gallbladder bile obtained from nine untreated cholesterol gallstone patients and eight cholesterol gallstone patients treated for 1 week with 600 mg/day of ursodeoxycholic acid. Bile obtained at cholecystectomy was ultracentrifuged for 2 h at 150,000 g to obtain isotropic samples. The conventional cholesterol crystal observation time was 3.1 +/- 4.1 (SD) days in controls and 19.0 +/- 1.9 days in the ursodeoxycholic acid-treated group (p < 0.001). Bile was analyzed by high-resolution gel-chromatography using 7 mM sodium taurocholate in the elution buffer. Biliary lipids eluted in four chromatographic zones: zone #I, corresponding to the column void volume, contained only minimal amounts of lipids; zone #II (apparent m.w. 100-220 kDa) comprised 29.1 +/- 12.4% of biliary cholesterol in the untreated group and 8.3 +/- 4.3% in the ursodeoxycholic acid-group (p < 0.001). At negative staining electron microscopy, this region was composed of roundish vesicles ranging from 7 to 20 nm in diameter. Zone #III (apparent m.w. 50-100 kDa) carried 59.1 +/- 2.1% of cholesterol in untreated patients and 81.2 +/- 9.5% in ursodeoxycholic acid-rich biles, respectively (p < 0.001). At negative staining electron microscopy, this region was composed of lamellar stacks of variable length, usually with 5 nm interspaces and up to 30 nm in width. In ursodeoxycholic acid-rich biles, lamellae often appeared in the form of concentric fingerprint-like images. Quasielastic light-scattering measurements in this region were compatible with the size estimates obtained at electron microscopy. Zone #IV (apparent m.w. 6-50 kDa) carried 11.8 +/- 9.4% and 11.6 +/- 9.0% of cholesterol, respectively (not significant). Since this region comprised a considerable fraction of endogenous bile salts and had no distinct morphological structures, it was interpreted as mixed micelles. The cholesterol crystal observation time showed a significant inverse correlation (r = -0.85, p < 0.001) with percent cholesterol carried by vesicles (zone #II) and a direct correlation (r = 0.86, p < 0.001) with percent cholesterol carried by lamellar bodies (zone #III). Vesicles and lamellae identical to those observed in isolated gel-chromatographic fractions were observed also on direct electron microscopic examination of unfractionated isotropic native biles. Similar findings were observed also in matched model biles.(ABSTRACT TRUNCATED AT 400 WORDS)