Prevention of retinoic acid-induced craniofacial abnormalities by folinic acid, and endothelin-1 (ET-1)/dHAND protein and mRNA expression were investigated in mouse embryos using the whole embryo culture, streptavidin-biotin peroxidase complex method, and whole-mount in situ hybridization. In the whole embryo culture, 1.0 and 0.1 mm-folinic acid dose dependently prevented branchial region malformations and decreased defects by 93 % and 77 %, respectively. Folinic acid at concentrations of 1.0 and 0.1 mm significantly increased ET-1 and dHAND protein expression levels compared to retinoic acid-exposed values in embryonic branchial areas. Folinic acid also increased ET-1 and dHAND mRNA levels in the same region. The present results suggest that folinic acid may prevent retinoic acid-induced craniofacial abnormalities via increasing ET-1 and dHAND levels in the branchial region during the organogenic period.