Massive apoptosis of pubertal male germ cells is important for the development of functional spermatogenesis in the adult testis. Although the trigger(s) for male germ cell loss at puberty remain undefined, we have hypothesized that transforming growth factor-betas (TGF-βs) play an active role. Here we demonstrate that the three mammalian TGF-β isoforms, TGF-β1, TGF-β2 and TGF-β3, induce distinct apoptosis of pubertal spermatogonia and spermatocytes in a dose-dependent manner. Induction of male germ cell death by activation of caspase-3 was most pronounced with TGF-β2 compared to TGF-β1 and TGF-β3. Furthermore, we found colocalization of activated caspase-3 with apoptotic protease-activating factor-1 (Apaf-1) in apoptotic germ cells, thus indicating the importance of the intrinsic mitochondrial pathway in TGF-β-induced apoptosis. The specificity of the TGF-β effects was proven by addition of recombinant latency-associated peptide against TGF-β1 (rLAP-TGF-β1) which completely abolished TGF-β1-induced and TGF-β3-induced germ cell apoptosis. Although TGF-β2-triggered germ cell death also was significantly reduced by rLAP-TGF-β1, inhibition was not maximal. Our results suggest that the three TGF-β isoforms induce apoptosis of pubertal male germ cells via the mitochondrial pathway in vitro and are thus likely candidates involved in the excessive first wave of apoptosis of male germ cells during puberty.