[Background] Embryonic development is coordinated by sets of cis-regulatory elements that are collectively responsible for the precise spatio-temporal organization of regulatory gene networks. There is little information on how these elements, which are often associated with highly conserved noncoding sequences, are combined to generate precise gene expression patterns in vertebrates. To address this issue, we have focused on Six3, an important regulator of vertebrate forebrain development.[Results] Using computational analysis and exploiting the diversity of teleost genomes, we identified a cluster of highly conserved noncoding sequences surrounding the Six3 gene. Transgenesis in medaka fish demonstrates that these sequences have enhancer, silencer, and silencer blocker activities that are differentially combined to control the entire distribution of Six3.[Conclusion] This report provides the first example of the precise regulatory code necessary for the expression of a vertebrate gene, and offers a unique framework for defining the interplay of trans-acting factors that control the evolutionary conserved use of Six3.This study was supported by grants from Spanish Ministerio de Educación y Ciencia (BFU-2004-01585) and in part by the EU (QLG3-CT-2001-01460) and the HFSPO (RGP0040/2001-M) to PB. A Telethon Foundation (GFP03007) and MEC (SB2003-0182) fellowships supported the postdoctoral work of IC.Peer reviewed