Abstract Rat testicular and adrenal gland microsomal preparations were incubated with 23,24-dinor-5-cholen-3β-ol (Guneribol) a proposed intermediate in the sesterterpene pathway for steroid biosynthesis. Steroids were isolated, purified by thin layer and high-performance liquid chromatography and crystallized to constant specific radioactivity. These preparations converted the substrate to 23,24-dinor-4-cholen-3-one. Radioactive 23,24-dinor-4-cholen-3-one was synthesized and incubated with further tissue preparations and shown to be converted to steroid hormones. These findings suggest that 23,24-dinor-4-cholen-3-one is an intermediate on the sesterterpene pathway for steroidogenesis.