OBJECTIVE: To establish a murine model of chemotherapy-induced diminished ovarian reserve (DOR) and investigate residual fertility after chemotherapy exposure. DESIGN: Two different chemotherapy protocols were tested to establish a valid DOR model by comparing follicle densities in mice given either protocol or physiological solution. An ovarian stimulation protocol was then selected from among different gonadotropins by counting the number of day 2 embryos obtained from normal mice. Finally, DOR mice were stimulated 5 and 8 weeks after chemotherapy with the chosen gonadotropin protocols, and day 2 embryos were recovered after mating, as was ovarian tissue for further immunohistologic analyses. SUBJECTS: Seventy-two Naval Medical Research Institute mice. EXPOSURE: Two different chemotherapy protocols. MAIN OUTCOME MEASURES: This study compared day 2 embryo counts in both normal and chemotherapy-induced DOR mice. Ovarian histology and morphology were also investigated by follicle counting and classification, as was immunostaining for apoptosis (cleaved caspase-3), activation (phospho-Akt), and proliferation (Ki67). RESULTS: A dose of 12 mg/kg of busulfan (Bu) + 120 mg/kg of cyclophosphamide (Cy) was chosen to establish the DOR model as it significantly reduced the ovarian reserve compared to both control mice (physiological solution) and the 1.2 mg/kg of Bu + 12 mg/kg of Cy protocol, without depleting it completely. When stimulated with 3.75 IU of Menopur, normal mice produced significantly more embryos than DOR mice given 12 mg/kg of Bu + 120 mg/kg of Cy (41.40 ± 14.74 vs. 23.67 ± 15.55 day 2 embryos). Although the follicle count was statistically diminished after single-dose chemotherapy administration, the remaining follicles did not display any difference in terms of apoptosis, activation, or proliferation rates. CONCLUSION: We successfully established a chemotherapy-induced DOR model using 12 mg/kg of Bu + 120 mg/kg of Cy, as evidenced by lower, but not completely depleted, follicle numbers and fewer retrieved embryos. Histologic study of ovarian tissue exposed to DOR-inducing chemotherapy revealed that surviving follicles were of the similar quality as tissue not exposed to chemotherapy.