Regulation of spermatogenesis involves stage-dependent androgen action on Sertoli cells, but the pathways involved are unclear. We assessed if cyclin D2 could play a role. In rats, Sertoli cell nuclear, stage-dependent immunoexpression of cyclin D2 switched on after Day 10 and persisted through Day 35, but disappeared by adulthood. However, ethane dimethane sulfonate (EDS)-induced testosterone withdrawal in adult rats for 6 days induced stage-dependent cyclin D2 immunoexpression in Sertoli cells, with highest expression at stages IX-XII and nondetectable at stages VI-VIII (opposite that for androgen receptor [AR] immunoexpression). In EDS-treated rats, a single injection of testosterone but not of estrogen reversed this change in 4 h, and testosterone administration from the time of EDS treatment prevented expression of cyclin D2 in Sertoli cells. The EDS-induced changes in cyclin D2 immunoexpression were matched by changes in expression of Ccnd2 (cyclin D2) mRNA in isolated stage-dissected tubules. Treatment of adult rats with flutamide induced stage-dependent cyclin D2 immunoexpression in Sertoli cells within 18 h, and confocal microscopy revealed that immunoexpression of AR and cyclin D2 were mutually exclusive within individual seminiferous tubules in these animals. Sertoli cell-selective ablation of the AR in mice using Cre/loxP technology also resulted in stage-dependent Sertoli cell cyclin D2 immunoexpression. Downstream from cyclin D2 action is retinoblastoma 1 (RB1), a tumor suppressor protein, immunoexpression of which paralleled stage-dependent AR expression in Sertoli cells; RB1 stage specificity disappeared after EDS treatment. These results point to a non-cell cycle role for cyclin D2 and RB1 in mature Sertoli cells in the stage-dependent mechanisms regulated by AR expression and androgen action.