The Sertoli cell ectoplasmic specialization is a unique junctional structure involved in the interaction between elongating spermatids and Sertoli cells. We have previously shown that suppression of testicular testosterone in adult rats by low-dose testosterone and estradiol (TE) treatment causes the premature detachment of step 8 round spermatids from the Sertoli cell. Because these detaching round spermatids would normally associate with the Sertoli cell via the ectoplasmic specialization, we hypothesized that ectoplasmic specializations would be absent in the seminiferous epithelium of TE-treated rats, and the lack of this junction would cause round spermatids to detach. In this study, we investigated Sertoli cell ectoplasmic specializations in normal and TE-treated rat testis using electron microscopy and localization of known ectoplasmic specialization-associated proteins (espin, actin, and vinculin) by immunocytochemistry and confocal microscopy. In TE-treated rats where round spermatid detachment was occurring, ectoplasmic specializations of normal morphology were observed opposite the remaining step 8 spermatids in the epithelium and, importantly, in the adluminal Sertoli cell cytoplasm during and after round spermatid detachment. When higher doses of testosterone were administered to promote the reattachment of all step 8 round spermatids, newly elongating spermatids associated with ectoplasmic specialization proteins within 2 days. We concluded that the Sertoli cell ectoplasmic specialization structure is qualitatively normal in TE-treated rats, and thus the absence of this structure is unlikely to be the cause of round spermatid detachment. We suggest that defects in adhesion molecules between round spermatids and Sertoli cells are likely to be involved in the testosterone-dependent detachment of round spermatids from the seminiferous epithelium.