Role of molecular oxygen in the course of the sidechain cleavage of progesterone by a microorganism, Cladosporium resinae was investigated with the double tracer method using 18O2 as a tracer of molecular oxygen, and 14C-labeled progesterone as a tracer of the substrate. Then, it was confirmed that, when progesterone was directly transformed to testosterone acetate by the microorganism, this enzymic reaction required molecular oxygen which was incorporated into the testosterone acetate as the oxygen atom located at 17β-position of testosterone. © 1968.