The present study was aimed at finding an effective method to isolate and purify the subtype of type A spermatogonial stem cells (SSCs) in juvenile rats. Testes from 9-days-old rats were used to isolate germ cells by using two-step enzymatic digestion. The expression of c-kit in the testes of the rats was immunohistochemically detected. After isolation, cell suspension was enriched further by discontinuous density gradient centrifugation. Then type A1–A4 spermatogonia was isolated from the purified spermatogonia with c-kit as the marker by using fluorescence-activated cell sorting (FACS). Electron microscopy was used to observe their ultrastructure. Finally, highly purified and viable subtype of SSCs was obtained. Cells separation with discontinuous density gradient centrifugation significantly increased the concentration of c-kit positive cells [(18.65±1.69)% after the centrifugation versus (3.16±0.84)% before the centrifugation, P<0.01]. Furthermore, the recovery and viability were also high [(65.9±1.24)% and (85.6±1.14)%]. It is concluded that FACS with c-kit as the marker in combination with discontinuous density gradient centrifugation can well enrich type A1–A4 spermatogonia from the testes of 9-days-old rats.