The expression domain of Otx2, a gene essential for the development of the fore- and midbrain, has previously been shown to be affected by exposure to all-trans-retinoic acid (AT-RA). However, morphological abnormalities of the fore- and midbrain induced by exposure of early somite-stage embryos to AT-RA were not associated with abnormal Otx2 expression. To identify abnormal expression of developmental genes induced by exposure at early somite-stages, we performed a fine analysis of the expression domains of Otx2, Otx1, Emx2, and Pax-6 by combining in situ hybridization (ISH) with computer-assisted superpositions and three-dimensional reconstructions of these expression domains. No alteration in the relative location of the caudal boundaries of the expression domains of these genes was observed. The only abnormality was a deletion of the most cranial portion of the neural folds (NF).