This study describes a method for isolating and characterizing various stages of spermatogenic cells from the prepuberal mouse testis. The authors define the morphological characteristics of different cell types and determine their temporal appearance during prepuberal development. They isolate seminiferous cords using collagenase and dissociate them with trypsin and DNase. The cells are then separated by sedimentation velocity at unit gravity, allowing for the recovery of specific cell types such as Sertoli cells, primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, leptotene/zygotene primary spermatocytes, pachytene primary spermatocytes, and Sertoli cells. The purity and identity of these cell types are verified using light and electron microscopy. The study also discusses the challenges and limitations of isolating early germ cells in the prepuberal testis due to the absence of spermatids. The successful isolation of these cell types provides a valuable resource for further biochemical and physiological studies on spermatogenesis.This study describes a method for isolating and characterizing various stages of spermatogenic cells from the prepuberal mouse testis. The authors define the morphological characteristics of different cell types and determine their temporal appearance during prepuberal development. They isolate seminiferous cords using collagenase and dissociate them with trypsin and DNase. The cells are then separated by sedimentation velocity at unit gravity, allowing for the recovery of specific cell types such as Sertoli cells, primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, leptotene/zygotene primary spermatocytes, pachytene primary spermatocytes, and Sertoli cells. The purity and identity of these cell types are verified using light and electron microscopy. The study also discusses the challenges and limitations of isolating early germ cells in the prepuberal testis due to the absence of spermatids. The successful isolation of these cell types provides a valuable resource for further biochemical and physiological studies on spermatogenesis.