Early in postnatal life, the first wave of spermatogenesis is accompanied by an initial wave of germ cell apoptosis. This may reflect an adjustment in the number of germ cells that can be adequately maintained by Sertoli cells. Two major pathways (intrinsic and extrinsic) are involved in the process of caspase activation and apoptosis in mammalian cells. The extrinsic pathway is characterized by the oligomerization of death receptors such as FAS or tumor necrosis factor, followed by the activation of caspase-8 and caspase-3. The intrinsic pathway involves the activation of procaspase-9, which in turn activates caspase-3. Extensive information is available concerning apoptotic inducers and their possible mechanisms in the adult rat. However, no data exist regarding the molecular and cellular mechanisms governing physiological cell death during puberty in the male rat. We have studied caspase activation throughout the first wave of spermatogenesis in the rat under physiological conditions, by combining the TUNEL procedure with the localization of active caspases in germ cells. We observed TUNEL-positive germ cells in rats of 5-40 days of age, the highest number being found in 25-day-old rats. TUNEL-positive and caspase-3-positive germ cells appeared as long chains of interconnected germ cells in 25-day-old rats. Caspase activation was assayed by either immunohistochemistry with antibodies against active caspase-3, -8, and -9, or by determining enzymatic activity in seminiferous tubules extracts. Both techniques showed activation of caspase-3, -8, and -9 in 25-day-old rats and low enzymatic activity at other ages. Confocal scanning laser microscopy indicated that active caspase-3, -8, and -9 co-localized with TUNEL-positive cells. Thus, caspase-3, -8, and -9 are active in apoptotic germ cells during the first wave of rat spermatogenesis. The extrinsic pathway of apoptosis may therefore play an important role in germ cell apoptosis during puberty in the rat.