We have developed a facilitated method for determining secretion of constituents into bile. The ratio of constituent/bilirubin was measured in gallbladder bile and multiplied by bilirubin secretion rate, estimated by measuring endogenous production of carbon monoxide (VCO) by breath sampling. Accuracy of this method was assessed by measuring secretion rate of 99mTechnetium-labeled disofenin during steady-state constant intravenous infusion. In nine subjects, mean (+/- SEM) secretion of disofenin by the CO method was 104.2 +/- 7.2% of expected and by standard marker perfusion was 97.8 +/- 13.1% of expected. In ten subjects, secretion rate of cholesterol by the CO method averaged 103 mumol/h by the CO method compared to 113 by marker perfusion (NS). Compared to marker perfusion (which is believed to reflect 24-h secretion rate), the CO method significantly underestimated secretion rate of bile acid (1110 vs. 1332 mumol/h, P = 0.076) and lecithin (295 vs. 413 mumol/h, P = 0.01), probably because gallbladder bile contained a disproportionate amount of fasting versus postprandial bile. Thus, this new method provides an accurate secretion rate for biliary constituents secreted at a relatively constant rate, including cholesterol, with less variability than marker perfusion. However, it can be used to estimate secretion of bile acid and lecithin only when a 20-30% underestimation of 24-h secretion is acceptable.