The Sertoli cell toxicant 2,5-hexanedione (2,5-HD) causes irreversible testicular atrophy in rats. After toxicant exposure, only Sertoli cells, stem cells, and a few spermatogonia remain in the seminiferous epithelium. In this study, the number, type, and fate of the remaining germ cells were determined. Male Sprague-Dawley rats were exposed to 1% 2,5-HD in drinking water for 5 weeks and then sacrificed 12 or 40 weeks after the start of exposure. Cell counts determined that the stem cell population was diminished in size, but made up a significant portion of the remaining germ cells. The remaining germ cells were primarily type A spermatogonia. Modeling of spermatogonial divisions suggested that most spermatogonia undergo degeneration at the level of type A3 spermatogonia after 2,5-HD-induced atrophy. Apoptosis was demonstrated to occur in the remaining germ cells by nuclear morphology and in situ analysis of DNA fragmentation. Quantitation indicated that apoptosis occurred in a majority of stem cell progeny. We conclude that the irreversibility of 2,5-HD-induced testicular injury results from the reduced size of the stem cell population as well as a block in germ cell development at the level of type A spermatogonia.