Testing for reproductive and developmental toxicity of drugsand other chemical compounds in vitro is an attractive alternativeprocedure to time-consuming and expensive in vivo orex vivo experiments. The embryonic stem cell test (EST) representsa scientifically validated method for the detection andclassification of compounds according to their teratogenic potency.However, more work is required to assess its applicabilitydomain and to improve its predictive capacity before gainingfull regulatory acceptance. We chose valproic acid (VPA) asa model compound to evaluate the suitability of the EST fordistinguishing between developmental toxicity potencies ofsubstances with closely related structures. VPA is among themost frequently used anti-epileptic drugs worldwide. Further, itis used for migraine prophylaxis and in the treatment of bipolarpsychotic disorders. Two severe side effects of VPA, hepatotoxicityand teratogenicity, have prompted research into derivativesof VPA. Here we investigate six closely related analoguesof VPA whose teratogenic potential has been previously determinedin the NMRI mouse model of encephalopathy. Distinctembryotoxicities in vivo of stereoisomers which differ only intheir spatial configuration were reproduced by the EST. Similarly,an increased potency in vivo correlating with longer chainlength of the congener was evident as higher toxicity in the EST.As toxicological endpoints, both differentiation and cytotoxicityin vitro have to be considered to assess teratogenicity comparableto in vivo results. In conclusion, our data demonstrate thatthe EST represents a valuable screening tool in potency rankingof structurally closely related substances of the same class.