Objective To isolate and purify Sertoli cells and spermatogonia from rat testis, and to study the proliferation and differentiation of the spermatogonia Cocultured with sertoli cells. BR> Methods After enzymatic digestions of rat testis, the suspension passed the BSA uncontinuous gradient medium in a gravity unit by velocity sedimentation. Then the spermatogonia were further purified by different times of attachment culture. Results The purities of Sertoli cells and spermatogonia were 92.73% and 78.36% respectively after velocity sedimentation separation. The semi-anchored spermatogonial cells were usually round or oval in outline, singly scattered or existed as aligned, clumped populations, while the outline of the attached Sertoli cells in plate was fibroblastlike cells with little protrusions. Conclusion The results suggest that the spermatogonial cells can survive and proliferate for some time in the culture medium containing EGF, bFGF and GDNF, while Sertoli cells can be more prolific and enhance the mitosis and proliferation of the spermatogonial cells. The confluent monolayer of Sertoli cells can be used as feeding layers for Coculture with spermatogonial