During fetal mouse development, germ cells enter the developing gonad at embryonic day (E) 10-11. In response to signaling from the male or female gonad, the germ cells commit either to spermatogenesis at E12.5 and enter mitotic arrest or to oogenesis and enter meiotic arrest at E13.5. It is unclear whether male commitment of the germ line and mitotic arrest are directly associated or whether they are developmentally separate. In addition, the published data describing the timing of mitotic arrest are inconsistent, and the molecular processes underlying the control of the cell cycle during mitotic arrest also remain unknown. Using flow cytometric techniques, 5-bromo-2'-deoxyuridine labeling, and immunofluorescent analysis of cell proliferation, we have determined that germ cells in the embryonic mouse testis arrest in G0 during E12.5 and E14.5. This process is gradual and occurs in an unsynchronized manner. We have also purified germ cells and analyzed molecular changes in male germ cells as they exit the cell cycle. This has allowed us to identify a series of molecular events, including activation of p27(Kip1), p15(INK4b), and p16(INK4a); the dephosphorylation and degradation of retinoblastoma protein; and the suppression of CyclinE, which lead to mitotic arrest. For the first time, the data presented here accurately define the mitotic arrest of male germ cells by directly combining the analysis of cell cycle changes with the examination of functionally defined cell cycle regulators.