1. The incorporation of [2-(14)C]acetate, [2-(14)C]mevalonic acid and [2-(14)C,2-(3)H(2)]-mevalonic acid into torulene and torularhodin by Rhodotorula rubra and Rhodotorula glutinis was studied. 2. A recovery of 14.3% of the label was obtained on decarboxylation of the torularhodin biosynthesized from [2-(14)C]mevalonic acid. 3. An analysis of the (3)H/(14)C ratio in torularhodin gave a value of 9.44:8. 4. These results, obtained by different experimental techniques, show that the reactions in the conversion of the dimethyl group of isopentenyl pyrophosphate into the 16',17'-position of torularhodin must be free from randomization. A mechanism for the isomerization of isopentenyl pyrophosphate to dimethylallyl pyrophosphate is suggested.