The antiepileptic drug valproic acid is a well-known teratogenic agent; its main target organ is the neuroepithelium neural tube, although skeletal malformations have also been described. The goal of our investigations was to determine whether there is a direct relationship between high doses of valproic acid and vertebral arch openings. On day 9 of gestation, rats were treated with either 0.3 ml physiologic saline or 600 mg/kg valproic acid (VA) given once in the morning and once in the evening (7 h between doses) for a total of 1200 mg/kg. For the teratological investigations, fetuses were examined at 21 days of gestation. Spina bifida occulta was demonstrated in double-stained fetal skeletons by measuring the distance between the cartilaginous ends of each vertebral arch. The most important finding was the very high rate of spina bifida occulta observed with this application regimen. Spina bifida aperta was not observed in our study. A low rate of exencephaly was observed in the treated group (3%). The difference between the control and treated groups was statistically highly significant from the first thoracic to fifth sacral level. The effects of VA are most pronounced from thoracic 9 to the upper lumbar region. The largest gap in vertebral arches was found in the first and second lumbar vertebrae in the VA-treated group. These findings suggest that the peak concentration of VA in the maternal plasma and high bolus administration of VA may be more important for spina bifida occulta than the total dose and infusion of VA.