Stem cells possess enormous therapeutic potential in tissue replacement. To study stem cells further, they must be isolated. Techniques are available for enrichment and study of hematopoietic stems cells, but thus far, techniques for purification of spermatogonial stem cells have not been described. Enrichment techniques for hematopoietic stem cells include the use of fluorescence-activated cell sorter analysis with Hoechst 33342 and rhodamine 123 (Rho) dyes. Use of Hoechst dye to isolate spermatogonial stem cells has been unsuccessful in our laboratory, and our results have conflicted with those from other laboratories. Taking advantage of the differential staining of the Rho dye, we report a novel method to enrich murine spermatogonial stem cells. Testicular cells are harvested from cryptorchid ROSA26 male mice. Populations of these cells are then stained with the Hoechst and Rho dyes, allowing them to be sorted by flow cytometry into a side population (SP) of Hoechst low-intensity cells and populations of low (Rho^low) or high (Rho^hi) fluorescent intensity. Sterile recipients, W/W^v mice, with an intrinsic germ cell deficiency were transplanted with the Hoechst SP cells, Rho^low, Rho^hi, and nonsorted donor cells. No spermatogonial stem cell colonies were derived from the Hoechst SP cells. The number of spermatogonial stem cell colonies from transplanted Rho^low cells showed a 17- and 20-fold enrichment over those of Rho^hi and nonsorted cells, respectively.