The fine structure of testicular interstitial cells of lion and three‐toed sloth showed an abundance of agranular endoplasmic reticulum, mitochondria and lipid droplets. Mitochondria were usually small and rod to rounded in shape, but large bizarre forms, such as triangular, oval and rounded with tubular and lamellar cristae were also observed. Mitochondria with lamellar cristae occurred in lion, but not in three‐toed sloth. These cristae were highly developed and appeared unique for the testicular interstitial cells. Both small and large mitochondria closely adhered to lipid droplets which were found singly or in clusters. The stored lipid droplets probably serve as a major source of substrate in androgen biosynthesis. The possible role of agranular endoplasmic reticulum in androgen biosynthesis is suggested.