CDB-4022 is under investigation as a potential oral male contraceptive. The goal of this study was to determine the acute effects of CDB-4022 on the ultrastructure of Sertoli and germ cells. Rats (4/time point) were treated orally with either vehicle or a maximally effective single dose of CDB-4022 (12.5 mg/kg) or Di-n-pentylphthalate (DPP; 2,200 mg/kg). Rats underwent perfusion fixation with 4% formaldehyde and 1% glutaraldehyde at 3, 6, or 12 hours postdosing for CDB-4022-treated rats, and 3 or 12 hours postdosing for vehicle or DPP-treated rats, respectively. Preserved testes were processed for transmission electron microscopy. Sertoli and germ cells of vehicle-treated rats demonstrated normal morphological characteristics. In contrast, disruption of Sertoli cell ultrastructure was readily apparent in CDB-4022-treated rats by 3 hours. A decrease in the presence of nucleoli, an increase in the amount and diameter of swollen smooth endoplasmic reticulum, and decreases in cytoplasmic ground substance were observed. Cytoplasmic rarefaction was observed in some Sertoli cells. The severity of these degenerative effects on Sertoli cells increased at 6 and 12 hours: Vacuoles were apparent in the cytoplasm; increased cellular debris, swollen mitochondria, and phagocytic structures were observed; and membranes became more disorganized. Similar ultrastructural changes were observed in the Sertoli cells of DPP-treated rats. By 3 hours post-dosing, spermatocytes and spermatids were also adversely affected by CDB-4022 treatment with swelling of the nuclear envelope such that two membranes were easily distinguished. The Step 8 spermatids were especially noteworthy because chromatin was more diffuse and rarefied, the nuclear envelopes were incomplete, broken, or missing portions, and the position of the spermatid nucleus within the cell and relative to Sertoli cell cytoplasm was unusual. Fusion of spermatids to form giant cells was also observed by 12 hours, but appeared to be secondary to retraction of Sertoli cell processes. These observations indicate that CDB-4022 acts acutely on Sertoli cells to induce marked cellular rarefaction and degeneration, but not necrosis. The data also suggest a rapid and direct effect of CDB-4022 on spermatocytes and spermatids. Collectively, the antispermatogenic activity of CDB-4022 appears to be a consequence of direct effects on both Sertoli and germ cells.