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Background & Aims: Many patients with cholesterol gallbladder stones (GBS) have a high percentage of deoxycholic acid (DCA) in gallbladder bile (all of which are in the conjugated form), probably as a result of prolonged large bowel transit times (LBTT). However, whether the prolonged LBTT increases DCA formation, solubilization, or absorption (or all 3) is not known. Methods: In 40 subjects (20 with GBS; age range, 24–74 years), we measured LBTT using radiopaque markers, and intestinal luminal pH by radiotelemetry. We also measured quantitative anaerobic bacteriology and the activities of 2 bile acid–metabolizing enzymes in fresh cecal aspirates obtained during clinically indicated unprepared colonoscopy, and related these results to the percentage of DCA in fasting serum measured by gas chromatography–mass spectrometry. Results: Compared with controls, GBS patients had longer LBTT (mean 23.1 ± SEM 2.8 h vs. 36.5 ± 3.3 h; P 9 vs. 5.9 ± 1.5 × 10 9 cfu/mL) and Gram-positive (9.5 ± 3.1 × 10 8 vs. 18.0 ± 4.1 × 10 8 cfu/mL; P −4 U/mg protein) in the cecal aspirates. They also had higher intracolonic pH values ( P P Conclusions: Slow colonic transit (more time), increased Gram-positive anaerobes (more bacteria), and greater 7α-DH activity (more enzyme) favor enhanced DCA formation; transit-induced increases in distal colonic luminal pH favor enhanced DCA solubilization/bioavailability; and increases in LBTT (more time) again favor DCA absorption. GASTROENTEROLOGY 2000;119:806-815