DNA-damaging cancer treatments cause female infertility and ovarian endocrine failure, due to depletion of the ovarian reserve. Attempts to address this have been hampered by limited understanding of the cellular mechanisms underlying follicle death after chemotherapy. We have previously reported that the pro-apoptotic BH3-only protein, PUMA, is the critical trigger of apoptosis in primordial follicles (PMFs) following cisplatin (Cis) or cyclophosphamide (Cy) treatment in mice. However, the specific targets of these drugs in the ovary, and the timing of follicular demise remain unclear. In this study we investigated the underlying processes leading to follicle death following these treatments. Adult wild-type mice were injected with saline (control), Cis 5 mg/kg, or Cy 300 mg/kg (N=5/group); ovaries were harvested after 8 hours, 24 hours, or 5 days. γH2AX immunofluorescence showed DNA double-stranded breaks (DSBs) in PMFs by 8 hours (saline-8h: 0% positive vs Cy-8h: 54±8% positive, p<0.01 vs Cis-8h: 38±8% positive, p<0.01). The proportion of γH2AX-positive follicles had reduced by 24 hours (saline-24h: 0% vs Cy-24h: 8%, NS vs Cis-24h: 16%, NS), and disappeared by 5 days (saline-5d: 0% vs Cy-5d: 8%, NS vs Cis-5d: 0%), indicating that PMF oocytes undergo either apoptosis or repair in this timeframe. Similar patterns of γH2AX positivity and disappearance were observed across all follicle stages. We then focused on the γH2AX-positive follicles to identify the cells targeted. In primordial, transitional, and primary follicles, only oocytes sustained DSBs, whereas in secondary and antral follicles, only somatic (granulosa ± theca) cells were affected. TUNEL staining further supported this conclusion. Given that 80% of oocytes are stored in PMFs in young adult mice, these data demonstrate that direct killing of PMF oocytes is the primary mechanism of ovarian reserve depletion caused by cisplatin and cyclophosphamide. Thus, future strategies to prevent chemotherapy-induced infertility must focus on preventing PMF oocyte death.