Six3 and Six6 are two genes required for the specification and proliferation of the eye field in vertebrate embryos,suggesting that they might be the functional counterparts of the Drosophila gene sine oculis (so) . Phylogenetic and functional analysis have however challenged this idea, raising the possibility that the molecular network in which Six3 and Six6 act may be different from that described for SO. To address this, we have performed yeast two-hybrid screens, using either Six3 or Six6 as a bait. In this paper,we report the results of the latter screen that led to the identification of TLE1 (a transcriptional repressor of the groucho family) and AES (a potential dominant negative form of TLE proteins) as cofactors for both SIX6 and SIX3. Biochemical and mutational analysis shows that the Six domain of both SIX3 and SIX6 strongly interact with the QD domain of TLE1 and AES, but that SIX3 also interacts with TLE proteins via the WDR domain. Tle1 and Aes are expressed in the developing eye of medaka fish( Oryzias latipes ) embryos, overlapping with the distribution of both Six3 and Six6 . Gain-of-function studies in medaka show a clear synergistic activity between SIX3/SIX6 and TLE1 ,which, on its own, can expand the eye field. Conversely, AES alone decreases the eye size and abrogates the phenotypic consequences of SIX3/6 over-expression. These data indicate that both Tle1 and Aes participate in the molecular network that control eye development and are consistent with the view that both Six3 and Six6 act in combination with either Tle1 and/or Aes.