Spermatogonial stem cells (SSCs) comprise a small population of germ cells that have self-renewal potential. However, studies on SSCs are hampered by the lack of SSC-specific markers. Although the cryptorchid operation is often used to obtain an enriched SSC population by destroying differentiating germ cells using high body temperature, SSCs in cryptorchid testes have different biological characteristics than those in a normal environment. Therefore, it is necessary to develop new methods for SSC selection. In this study, we report a method of isolating SSCs from wild-type mouse testes based on their functional characteristics using aldehyde dehydrogenase (ALDH) activity levels, which have been successfully used for stem cell isolation from many self-renewing tissues. Testis cells selected using CD9 or CDH1, both of which are expressed by SSCs, exhibit ALDH activity in flow cytometric analyses. However, spermatogonial transplantation revealed that SSCs do not show ALDH activity, whereas somatic stem cells have high ALDH activity levels. Nevertheless, SSCs could be enriched 183.7-fold based on CDH1 preselection and transplantation of cells that lacked ALDH activity. In contrast, we failed to enrich SSCs from cultured spermatogonia, which exhibited ALDH upregulation in vitro. These results suggest that SSCs are unique among tissue-specific stem cells in their regulation of ALDH activity. Development of a new technique for SSC isolation from wild-type testes based on their functional properties will facilitate investigation of SSCs in a normal testicular environment.