The neurotoxic hexacarbon 2,5-hexanedione (2,5-HD) produces testicular atrophy in experimental animals. To examine the morphogenesis of the testicular lesion, 1.0% 2,5-HD was provided in the drinking water of adult F-344 rats for up to 6 weeks. After 3 weeks of administration, there were occasional large vacuoles in the basal region of the germinal epithelium. At 4 weeks, these vacuoles were much larger and more numerous; electron microscopy demonstrated that they were derived from the smooth endoplasmic reticulum. The vacuoles were preferentially associated with stages 12, 13, 14, and 1 of the spermatogenic cycle. Additionally, at 4 weeks there was a significant decrease in the number of tubules in stages 7 and 13, and a concomitant increase in the percentage of tubules in stages 3, 5, and 6. By Week 5, most Golgi-phase and cap-phase spermatids were visibly affected, showing margination of nuclear chromatin, and were becoming dissociated from Sertoli cells. Frequent multinucleated giant cells were seen and electron microscopy of these cells suggested that they were derived from fused spermatocytes or spermatids. After 6 weeks, fewer giant cells were present, most tubules contained cellular debris, and many showed empty lumina encircled by a thin ring of cytoplasm near the basement membrane. Interstitial tissue appeared unaffected. These studies indicate that the Sertoli cell is probably an initial target for 2,5-HD action in the testis.