The effects of a four to six day fast on gallbladder bile lipid composition, bile acid pool size, bile acid composition, and cholic acid metabolism have been determined in normal human subjects. Total bile acid pool size and cholic acid pool size were measured before and after fasting by a one-sample technique previously validated in our laboratory. The rate of synthesis of cholic acid and its fractional turnover rate before fasting were measured using standard techniques. Estimates of fasting cholic acid synthesis rate and fractional turnover rate were calculated as daily averages from the change in cholic acid pool size, in combination with the change in cholic acid specific activity, during the fasting period. Since these estimates are approximate, a maximum value for cholic acid synthesis rate during fasting was also determined by assuming that the entire change in cholic acid specific activity during the fasting period occurred instantaneously. The molar percent of cholesterol in gallbladder bile was reduced in eight of nine subjects after a four to six day fast (p less than .01; mean reduction 30.5%). The molar percents of bile acid and phospholipid were not significantly altered by fasting. The cholesterol saturation index, calculated on the basis of these data, was reduced by an average of 31.0% after a four to six day fast (p less than .02). The average daily cholic acid synthesis rate and the fractional turnover rate were reduced in all six subjects on whom isotope kinetic studies were carried out. The mean decrease in synthesis rate was 68.5% (p less than .05; range 55.2-79.8%) while the mean decrease in fractional turnover rate was 64.4% (p less than .05; range 30.2-100%). Reduction in synthesis rate was confirmed by the determination of maximum fasting synthesis of cholic acid, which averaged 61.1% lower than synthesis in the fed period. Fasting had no consistent effect on total bile acid pool size, cholic acid pool size, or bile acid species composition.