The use of FEO as a remedy for control of primary dysmenorrhea increases concern about its potential teratogenicity due to its estrogen-like activity. Limb bud mesenchymal cells, when grown in high-density cultures, can be differentiated into a number of cell types including cartilage and muscle. These cells have been used extensively for in vitro studies of chondrogenesis. Therefore, we used limb bud cells and Alcian blue staining method that is specific for staining cartilage proteoglycan, to determine the teratogenic effect of FEO. Limb bud cells obtained from day 13 rat embryo were cultivated and exposed to various concentrations of FEO for 5 days at 37 degreesC and the number of differentiated foci were counted. Retinoic acid (90 mug/ml) was chosen as positive standard control. The differentiation was also evaluated using limb bud micromass culture using immunocytochemical techniques and BMP-4 antibody. The results showed that FEO at concentration as low as 0.93 mg/ml produced a significant reduction in the number of stained differentiated foci. However, this reduction was due to cell loss, determined by neutral red cell viability assay, rather than to be related to decrease in cell differentiation. These findings suggest that the FEO at the studied concentrations may have toxic effect on fetal cells, but there was no evidence of teratogenicity. (C) 2004 Elsevier Ltd. All rights reserved.