Although it is likely that a highly complex network of transcription factors acts in concert during mammalian brain development, relatively few such genes have been characterized to date. We describe here a novel murine homeobox gene, denoted Gtx, which in adult animals is specifically expressed within glial cells of the central nervous system, including the forebrain, and in germ cells of the testis. Gtx resides on chromosome 7 and does not cosegregate with any previously mapped homeobox gene. The amino acid sequence of the predicted protein encoded by Gtx is highly divergent from that of any other known homeobox genes. The Gtx homeodomain contains unique residues at positions predicted to contact DNA bases. It did not bind to known target sites for other homeobox genes in vitro but bound with high affinity to the MEF-2 motif, a binding site for the serum response factor-related proteins. GTX efficiently competed with RSRF to bind the MEF-2 element in vitro. Co-transfection of Gtx prevented the serum-induced activation of the MEF-2-containing reporter genes. Although the true biological role of Gtx is not known, these results suggest that Gtx is a novel cell-type specific homeobox gene that has the potential to act as a transcriptional repressor for a subset of serum-inducible genes.