Testosterone is the primary androgen driving male sexual differentiation and masculinization and supporting spermatogenesis in mammalian species. Deficiencies in androgen biosynthesis during fetal development, although rare, can lead to developmental defects. Testosterone is synthesized by the Leydig cells of the testis from cholesterol that is transferred from intracellular stores into Leydig cell mitochondria. Cholesterol translocation is the hormone-sensitive and rate-limiting step in androgen biosynthesis. This process is achieved through a hormone-dependent reorganization of organelles and by protein and lipid interactions. After translocation into the mitochondria, cholesterol is metabolized by cytochrome CYP11A1 into pregnenolone, which is further metabolized to androgens in the smooth endoplasmic reticulum by the steroidogenic enzymes 3β-HSD, CYP17A1, and 17β-HSD3. Steroidogenesis is sustained by a continuous supply of either de novo synthesized cholesterol or cholesterol coming from lipoproteins. Adult Leydig cells produce testosterone in response to luteinizing hormone (LH) produced by the pituitary in response to gonadotropin-releasing hormone (GnRH) from the hypothalamus. Serum testosterone levels are reduced with aging. Age-related decreases in testosterone may result from reduced LH and/or from the reduced responsiveness of the Leydig cells to LH, resulting in reduced cholesterol availability to CYP11A1. Age-dependent reduced Leydig cell responsiveness to LH has been shown to be due to oxidant/antioxidant imbalance in the Leydig cells. The cellular mechanisms underlying testosterone formation and its regulation by LH in the adult and aged Leydig cells are discussed.