The goal of this study was to develop a method for ultrasound-guided percutaneous intraovarian injection in Wistar rats. Intraovarian administration of chemicals or needle aspiration of the ovary has been undertaken in some species, including humans, equines, and bovines. In rodents, which are widely used in scientific research, a technique for intraovarian injection without surgical exposure of the organ has not been described. The current study standardized the procedure of ovarian percutaneous injection of 0.9% sodium chloride guided by ultrasound in rats. The ovaries were measured by ultrasound imaging before and immediately after injection and showed a significant increase in ovarian length but not width. No clinical abnormalities were detected within 15 d after injection. These findings indicate that the steps of ultrasound localization of the organ, digital restraint, and correct needle insertion achieved successful intraovarian administration of saline without invasive surgery. These results document the feasibility of ultrasound-guided intraovarian percutaneous injection in rats and may be useful for future research on female reproduction and chemical sterilization.