Folic acid deficiency during conception up to the end of the third month of gestation is believed to play the most important factor in neural tube defects (NTDs). However, the exact molecular mechanism remains to be elucidated. It has been suggested that transforming growth factor-beta (TGF-beta1) and insulin-like growth factor-1 (IGF-1) play a critical role in supporting bone formation. Therefore, folic acid deficiency may contribute to NTD occurrence via decreased TGF-beta1 and IGF-1 expression. This study aimed to determine the correlation between folic acid deficiency and the expression of TGF-beta1 and IGF-1 in rat skull bone. Thirty female Sprague-Dawley rats were divided into three groups. Purified diet containing 5 (restricted), 15 (low) and 30 microg (normal) of folic acid was given to the first, second and third groups, respectively. At 16 weeks of a given diet, blood samples were taken to examine folic acid (folate immunoassay method), TGF-beta1 and IGF-1 (enzyme-linked immunosorbent assay method) levels. After forced mating, on the 18th-19th day of gestation (E18-19), the pregnant rats were subjected to hysterectomy. The skull bone samples of E18-19 rats were taken to examine the TGF-beta1 and IGF-1 protein expression by immunohistochemistry. The folic acid-restricted diet (5 microg) resulted in decreased serum TGF-beta1 and IGF-1 levels. Furthermore, protein expression of TGF-beta1 and IGF-1 in E18-19 rat skull bones was also significantly lower in the folic acid-restricted diet than in the normal diet. Folic acid deficiency could result in reduction of TGF-beta1 and IGF-1 protein levels and might contribute to formation of defects in the skull bone as observed in mengingocele patients.