Objective: To examine the effects of lipiodol, melatonin, and radiation used during hysterosalpingography (HSG) on rat ovarian follicle reserve. Materials and Methods: A total of 50 Wistar rats with regular estrous cycles were randomly divided into five groups. Group 1 was the control group. The other groups, X-ray was applied (G2), 0.1 ml lipiodol was applied to each uterine horn (G3), 20 mg/kg intraperitoneal melatonin application was followed by 0.1 ml lipiodol administration to each uterine horn after 15 minutes (G4), 20 mg/kg melatonin was administered to ligamentum suspensorium ovarii, followed by 0.1 ml lipiodol application to each uterine horn after 15 minutes (G5), respectively. The rats in G2, G3, G4, and G5 were exposed three times to whole body radiation. Then, all rats were re-opened and left oophorectomy was performed. Left ovarian samples were fixed in formaldehyde. Primordial, primary, secondary, and tertiary follicles were counted in the preparations, and all were added to calculate the ovarian follicle reserve. Results: Primordial, primary, and ovarian follicle reserves were found significantly lower in G2, in comparison to other groups. Atretic follicle values were significantly higher in G2 compared to G3, and in G3 compared to the other groups. Regression of angiogenesis within corpus luteum was found significantly lower in G2, compared to G3, and in G3 compared to other groups. All values in G1, G4, and G5 were similar. Conclusion: Use of lipiodol and melatonin during HSG procedure prevents the negative effects of radiation on ovarian follicle reserve.