A transverse vaginal septum was found in 2% of a substrain of specific-pathogen-free Wistar rats used in reproductive toxicity studies. Macroscopic observation mostly revealed a complete transverse septum. In a few rats an incomplete septum with a small central pupillary gap was found. Microscopically the septum consisted of a central core of connective tissue covered on both sides by epithelium. The rats with vaginal defects were clinically healthy but were unable to breed because the vaginal septum hampered the progress of the spermatozoa. Cranial to the defect, the uterus, cervix, and cranial portion of the vagina were moderately to severely dilated and often filled with mucopurulent fluid and cellular debris. The presence of a transverse vaginal septum is rare in rats and can therefore be easily missed. Congenital, embryologic, and histologic aspects of this anomaly are discussed and compared with existing literature data.