The Hox-1.6 gene disrupted in embryonic stem cells by homologous recombination was introduced into the mouse germline. Heterozygous mice were normal, but homozygous mice died at birth from anoxia and had numerous defects that were centered at the level of rhombomeres 4 to 7 and included delayed hindbrain neural tube closure, absence of certain cranial nerves and ganglia, and malformed inner ears and bones of the skull. Thus, Hox-1.6 is involved in regional specification along the rostrocaudal axis, but only in its most rostral domain of expression. Hox-1.6 appears to specify neurogenic neural crest cells prior to specification of mesenchymal neural crest cells by Hox-1.5. Thus, within the same region of the presumptive hindbrain, two HOX-1 genes are involved in the patterning of two different populations of neural crest cells. The implication of these results for the function of the Hox network during mouse embryogenesis is discussed.