Involvement of the Hox genes in regional specifications of the vertebrate body axis is suggested by sequence similarity with the homeotic selector genes of Drosophila, the conservation of a collinear relationship between genomic organization and site of expression, and mutational analysis. Subdivision of vertebrate embryo hindbrain neuroepithelium into lineage compartments (rhombomeres) underlies segmental patterning of neuronal differentiation. The rhombomere boundaries delimit domains of expression of Hox genes, presumed to be determinants of rhombomere phenotype, suggesting that Hox genes confer positional value; the formation of rhombomere 4 (r4) is followed by strong expression of Hox-2.9 within its confines. If the Hox genes are determinants, their expression should be autonomous from the developmental stage at which regional commitment becomes fixed and irreversible. We have transplanted the future r4 region (from state-9-chick embryos) into the more anterior position of r2 and probed for Hox-2.9 transcripts. We report here that Hox-2.9 was expressed in the ectopic r4 as strongly as in the normal r4, whereas reciprocal grafts of future r2 to r4 position did not express Hox-2.9. The phenotype of ectopic rhombomeres developed according to their original position, as demonstrated by retrograde tracing of efferent cranial nerve nuclei. As early as stage-9-(six somites), both Hox-2.9 expression and segment identity are autonomous in the chick embryo hindbrain, independent both of position in the neuroepithelium and of signals from the underlying mesoderm.