By in situ hybridization of quail neuroretinas, we observed that Engrailed (En-1) is expressed both in the ganglionic and the amacrine cell layers, similarly to Pax-6. Because we observed a decrease of Pax-6 expression in the neuroretina of hatched animals, we studied the effect of the chicken En-1 and En-2 proteins on Pax-6 expression. En-1 and to some extent En-2 were able to repress the basal and the p46Pax-6-activated transcription from the two Pax-6 promoters. Infection of retinal pigmented epithelium by a virus encoding the En-1 protein repressed the endogenous Pax-6, and a similar effect was observed with a homeodomain-deleted En-1. In vitro interaction indicates that En proteins are able to interact with the p46Pax-6 through the paired domain. This interaction negatively regulates the DNA-binding properties of the p46Pax-6. These results suggest an interplay between En-1 and Pax-6 during the central nervous system development and indicate that En-1 may be a negative regulator of Pax-6.