We performed studies on the mechanisms for the teratogenicity of trypan blue (TB) that is known as a potent teratogenic dye in rodents. TB was administered to Wistar rats at various doses and at different stages of gestation. Teratogenicity and embryolethality were observed by a single (50 and 250 mg/kg) subcutaneous injection and three (25 and 50 mg/kg) subcutaneous injections of TB daily from day 7 of pregnancy. The incidences of malformations in groups given 50 and 250 mg/kg on day 7 were 12 and 59%, respectively. Most of the fetuses with external malformations were accompanied with skeletal and/or internal anomalies. The types of frequently occurring malformations were as follows: exencephaly, spina bifida, tail anomaly, vertebral deformity, hydrocephaly and heart anomaly. Fetal toxicity was decreased after treatment with the mixture of TB and normal rat serum. The serum level of TB in pregnant rats increased to 308 micrograms/ml at one hour after subcutaneous treatment with TB 50 mg/kg and decreased rapidly, but remained at 58 micrograms/ml 72 hours later. Lower serum levels of TB were observed in pregnant rats given TB with serum. No fetotoxic effects of serum from pregnant rats treated with TB were observed in recipient rats given the serum on day 7, 8 and 9 of pregnancy.