Regulation of bile acid synthesis in man is incompletely understood, in part because of difficulty in making measurements over short time periods when the enterohepatic circulation is intact. We investigated the possibility of a diurnal rhythm of bile acid synthesis in three human subjects given [26-14C]cholesterol. When this isotope of cholesterol, which is randomly labeled in the 26 and 27 positions, is converted to bile acid, the 14C is released as propionic acid randomly labeled in the 1 and 3 positions. The labeled propionic acid is then oxidized to 14CO2, output of which is a function of bile acid synthesis. However, delays in transit of the 14C through propionic acid and CO2-HCO-3 pools would shift the phase and dampen the amplitude of 14CO2 output relative to an existing diurnal rhythm of bile acid synthesis. Therefore, using constant infusion methods, we determined the turnover constants for conversion to 14CO2 of [1-14C]propionic acid and [3-14C]propionic acid to be 0.36-0.59 h-1 and 0.14-0.16 h-1, respectively. Using these constants and modeling the diurnal rhythm as a cosine function, we determined that amplitude of 14CO2 output from [26-14C]cholesterol was reduced 35% and acrophase was delayed 2.4-3.0 h relative to the diurnal rhythm of bile acid synthesis. None of the diurnal rhythm in 14CO2 output from [26-14C]cholesterol resulted from diurnal variation in propionic acid or CO2-HCO-3 metabolism since constant infusion of [1-14C]propionic acid and [3-14C]propionic acid for 30 h revealed no diurnal variation in output of 14CO2. These studies demonstrate for the first time that humans with an intact enterohepatic circulation have a diurnal rhythm of bile acid synthesis with an amplitude of +/- 35-55% around mean synthesis, and an acrophase at about 9 a.m.