Two mouse genes, En-1 and En-2, have sequence homology to the engrailed (en) and invected (inv) genes of Drosophila (Joyner et al. 1985). Partial nucleotide sequence analyses of genomic and cDNA clones show that the homologous sequences can encode a stretch of 107 amino acids, including a centrally located, 60-amino-acid homeo box. Within the homologous regions of the mouse and Drosophila genes, 78 (73%) of the amino acids are identical. Such extensive conservation of sequence outside the homeo box between vertebrate and Drosophila homeo box-containing genes is thus far unique. En-1 and En-2 are expressed during mouse embryogenesis. Transcripts from both genes were detected in RNA samples from teratocarcinoma cells, which serve as in vitro models for the early embryo, and from embryos at 9.5 through 17.5 days of development. For each gene we observed a unique pattern of changes in the number and relative intensities of transcripts detectable during embryonic development. Transcripts from both genes are represented abundantly in RNA extracted from the posterior portion of the fetal brain, and are much less abundant in RNA from other fetal tissues, including the anterior portion of the brain and the spinal cord. The chromosome map positions of En-1 and En-2 were determined by recombinant inbred strain analyses. Unlike their Drosophila counterparts, they are unlinked: En-1 is in the central portion of the mouse chromosome 1 and En-2 is in the proximal portion of mouse chromosome 5. Both genes map in the vicinity of mutations that are known to cause abnormalities during development.