Abstract: The germ cell suspension was obtained by twostep enzymatic digestion from 5monthold calves testes.Spermatogonia were isolated and purified using Percoll discontinue density gradient centrifugation.Cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum(FBS) at 37 ℃, in a humidified atmosphere with 5% CO2. Growth and morphologic changes of cells were observed. Results: seminiferous tubules of 5monthold calf testis were mainly composed of spermatogonia and Sertoli cells, 3.18×106 cells were available per 1.0 gram testis parenchyma. The purity of the spermatogonia was up to 69.27%. Spermatogonia mainly distributed in 27%-35% Percoll gradient. The bovine spermatogonial stem cells began to divide after 6-7days culture and clones were formed after 20 days. Conclusion: The spermatogonia obtained by twostep enzymatic digestion and Percoll discontinue density gradient centrifugation could satisfy the needs of survival and proliferation of bovine spermatogonia in vitro.