FourDlxhomeobox genes,Dlx1,Dlx2,Dlx5, andDlx6are expressed in the same primordia of the mouse forebrain with temporally overlapping patterns. The four genes are organized as two tail-to-tail pairs,Dlx1/Dlx2andDlx5/Dlx6, a genomic arrangement conserved in distantly related vertebrates like zebrafish. TheDlx5/Dlx6intergenic region contains two sequences of a few hundred base pairs, remarkably well conserved between mouse and zebrafish. Reporter transgenes containing these two sequences are expressed in the forebrain of transgenic mice and zebrafish with patterns highly similar to endogenousDlx5andDlx6expression. The activity of the transgene is drastically reduced in mouse mutants lacking bothDlx1andDlx2,consistent with the decrease in endogenousDlx5andDlx6expression. These results suggest that cross-regulation by Dlx proteins, mediated by the intergenic sequences, is essential forDlx5andDlx6expression in the forebrain. This hypothesis is supported by cotransfection and DNA-protein binding experiments. We propose that theDlxgenes are part of a highly conserved developmental pathway that regulates forebrain development.