Cranial sense organs and a subset of cranial sensory neurons are generated from placodes, thickenings of the ectoderm. Pax3 has been known as a marker for ophthalmic trigeminal placode specification, and also an important regulator of trigeminal placode neuron differentiation. In this study, I show that Pax6 is initially expressed in the preplacodal region at the level of ophthalmic trigeminal placode, and that this expression gradually regresses in a medial-to-lateral direction as Pax3 expression expands in the same direction. Misexpression studies revealed that Pax6 represses Pax3 expression indirectly as a transcriptional activator in a cell-autonomous manner. Pax3-misexpression represses Pax6 expression in an indirect fashion, suggesting that unknown factor(s) downstream of Pax3 may repress Pax6 expression, and thereby allow an expansion of Pax3-positive ophthalmic trigeminal placode region. These results indicate that the mutual repression between Pax3 and Pax6 has important roles in the specification and the positioning of the ophthalmic trigeminal placode.