The roe deer (Capreolus capreolus) is a seasonal breeder. The cyclic changes between totally arrested and highly activated spermatogenesis offer an ideal model to study basic mechanisms of spermatogenesis. In this study, we demonstrated, to our knowledge for the first time, c-kit receptor-positive cells in the testis of roe deer. They were immunohistologically identified mainly as spermatogonia. Analysis of the amount of those cells by flow cytometry shows a distinct seasonal pattern, with pronounced differences between cells in the diploid state and in the G2/M phase of mitosis. The specific seasonal pattern of spermatogonial proliferation results in the increased relative abundance of spermatogonia as well as in their increased total number per testis in November and December. This suggests that cell divisions continue on a level sufficient to accumulate spermatogonia during winter. The serum concentrations of LH and FSH showed a peak in spring; testosterone showed a maximum concentration during the rut (July/August). The peak of both gonadotropins seems to precede the period of stimulated spermatogonial proliferation in spring. The testosterone peak coincides with maximal meiotic intensity in August. The results suggest the importance of testosterone for sperm production, and they provide a basis for detailed investigations of regulatory factors of the proliferation of spermatogonia.