During biosynthesis of bile acid, carbons 25–26–27 are removed from the cholesterol side chain. Side‐chain oxidation begins either with hydroxylation at the 26‐position, in which case the three‐carbon fragment is released as propionic acid, or with hydroxylation at the 25‐position, in which case the three‐carbon fragment is released as acetone. In the present study, we have quantitated the relative importance of these two pathways in vivo by measuring production of [14C]acetone from [14C] ‐26‐cholesterol. Four days after intraperitoneal injection of 20 to 40 μCi [14C]‐26‐cholesterol and 1 day after beginning a constant intravenous infusion of unlabeled acetone at 25 μmoles per kg per min, 6 male and 2 female Sprague‐Dawley rats underwent breath collections. Expired acetone was trapped and purified as the 2,4‐dinitrophenylhydrazine derivative. 14CO2 was trapped quantitatively using phenethylamine. Specific activity of breath acetone was multiplied times the acetone infusion rate to calculate production of [14C]acetone. [14C] Acetone production averaged 1.7% of total release of 14C from [14C]‐26‐cholesterol, estimated by 14CO2 output. The method was validated by showing that [14C] acetone production from [14C]isopropanol averaged 111% of the [14C]isopropanol infusion rate. We conclude that, in the normal rat, the 25‐hydroxylation pathway accounts for less than 2% of bile acid synthesis.