Exposure of male rats to cyclophosphamide, a commonly used anticancer and immunosuppressive drug, has been shown to alter fertility and progeny outcome in a male germ cell phase-specific manner. The effect of toxicant exposure on male germ cells depends in part on the stress response mechanisms present during the different stages of spermatogenesis. To assess how acute cyclophosphamide exposure affects the expression of stress response genes, we examined the expression of 216 genes, using gene expression arrays, in isolated rat spermatogenic cell types (pachytene spermatocytes, round spermatids, and elongating spermatids). Cyclophosphamide exposure affected gene expression in all cell types but most dramatically in round spermatids. Increased transcript levels were observed for 30 genes in round spermatids compared to seven genes in pachytene spermatocytes and two in elongating spermatids. The expression of genes involved in apoptosis, DNA-damage recognition and repair, transcriptional activation, and in the heat shock protein-chaperone response was most affected by cyclophosphamide in round spermatids. Our results demonstrate that cyclophosphamide alters the expression of stress response genes during spermatogenesis in a germ cell-specific manner. The greater response of round spermatids to cyclophosphamide suggests that this cell type may be more susceptible to the damaging effects induced by this drug, possibly due to the chromatin remodeling that is taking place at this stage of spermatogenesis. This observation is consistent with the reported higher level of abnormal progeny outcome seen when the germ cells were first exposed to cyclophosphamide as round spermatids.