Six rat homeo-box-containing DNA sequences have been isolated by screening a genomic library with a probe derived from an Antennapedia (Antp) cDNA clone of Drosophila melanogaster. Sequence determination of two of the clones containing the homeo-box regions reveals a 180-nucleotide(nt) domain sharing more than 80% homology at the nucleotide level and more than 90% homology at the amino acid level with the homeo-box from the Antp gene and from homeo-boxes of other metazoan species. Genomic blotting experiments suggest that the two homeo-box-containing DNA regions are present in one or two copies per haploid rat genome. Northern blot analysis of RNA has shown that the rat homeo-box sequences are expressed in a tissue-specific manner; transcripts were detected in the spinal cord and kidney, but not in brain, testis, liver, and spleen. The rat nucleotide sequences lying outside the 180-nt homeo-box domain share virtually no sequence homology with the Antp flanking regions. However, one clone does show an equally high degree of amino acid homology within the homeo-box and its immediate flanking region with a putative homologous gene in mouse. The result suggests that some of the mammalian homeo-box-containing genes are conserved in evolution and may serve important cellular or developmental functions.