Male rats treated with cyclophosphamide, an alkylating agent commonly used clinically in both acute and chronic regimens, present with damaged male germ cells and abnormal progeny outcome. The extent and type of damage induced by cyclophosphamide largely depend on the germ cell type exposed to the drug and its ability to respond to insult. In the present study, the response of pachytene spermatocytes to damage was evaluated by assessing their ability to undergo meiotic G₂/MI transition following exposure to acute or chronic cyclophosphamide. Male rats were given an acute high dose (70 mg/kg, once) or chronic low doses (6 mg/kg, daily for 5–6 wk) of cyclophosphamide. Pachytene spermatocytes were isolated, cultured, and induced to undergo G₂/MI transition with okadaic acid. To determine the effect of DNA damage on meiotic progression, induction of DNA double-strand breaks was detected after each treatment regimen by the formation of foci of phosphorylated histone H2AX. The transition from G₂ to MI was impaired after acute cyclophosphamide treatment; this impairment in the progression of pachytene spermatocytes was correlated with extensive DNA double-strand breaks. In contrast, despite the presence of significant levels of DNA damage, meiotic progression was not impaired in spermatocytes after chronic cyclophosphamide exposure. We suggest that the cell cycle impairment induced after acute cyclophosphamide treatment could be mediated by a G₂/M checkpoint activated in response to DNA damage. The absence of impairment after chronic treatment raises concern about the functionality of defense mechanisms in male germ cells after repeated exposure to low doses of genotoxic agents.