Disulfiram causes undulation of the notochord and bending of pharyngeal cartilages in fish embryos. Using flounder embryos, this study aimed to elucidate which process of pharyngeal arch development was affected by the drug. Since disulfiram is known to block the synthesis of retinoic acid (RA) in vivo, we first examined whether the drug suppresses the expression of Hoxd-4 and shh, RA responsive genes related with the pharyngeal arch development. Disulfiram at a concentration which induces undulation of the notochord and bending of cartilage elements did not affect the expression of these genes. On the other hand, similar phenotypes of anomalies were found to be reproduced both in the notochord and pharyngeal cartilages by α, α′-dipyridyl which reduces the mechanical stability of collagen. Thus, we suppose that disulfiram causes anomalies by decreasing the mechanical stability of collagen and not by suppressing the expression of RA-responsive genes. Since disulfiram blocks ascorbate dehydrogenase, it is our hypothesis that this drug inhibits the maturation of collagen by affecting ascorbic acid metabolism.