Using immunohistochemistry, the expression of the D-type cyclin proteins was studied in the developing and adult mouse testis. Both during testicular development and in adult testis, cyclin D₁ is expressed only in proliferating gonocytes and spermatogonia, indicating a role for cyclin D₁ in spermatogonial proliferation, in particular during the G₁/S phase transition. Cyclin D₂ is first expressed at the start of spermatogenesis when gonocytes produce A₁ spermatogonia. In the adult testis, cyclin D₂ is expressed in spermatogonia around stage VIII of the seminiferous epithelium when A_al spermatogonia differentiate into A₁ spermatogonia and also in spermatocytes and spermatids. To further elucidate the role of cyclin D₂ during spermatogenesis, cyclin D₂ expression was studied in vitamin A-deficient testis. Cyclin D₂ was not expressed in the undifferentiated A spermatogonia in vitamin A-deficient testis but was strongly induced in these cells after the induction of differentiation of most of these cells into A₁ spermatogonia by administration of retinoic acid. Overall, cyclin D₂ seems to play a role at the crucial differentiation step of undifferentiated spermatogonia into A₁ spermatogonia. Cyclin D₃ is expressed in both proliferating and quiescent gonocytes during testis development. Cyclin D₃ expression was found in terminally differentiated Sertoli cells, in Leydig cells, and in spermatogonia in adult testis. Hence, although cyclin D₃ may control G₁/S transition in spermatogonia, it probably has a different role in Sertoli and Leydig cells. In conclusion, the three D-type cyclins are differentially expressed during spermatogenesis. In spermatogonia, cyclins D₁ and D₃ seem to be involved in cell cycle regulation, whereas cyclin D₂ likely has a role in spermatogonial differentiation.