Mice carrying a nonfunctional allele of hoxc-4 have been generated by gene targeting. The phenotype of mice homozygous for this mutation is strikingly different from those reported in mice lacking the paralogous genes hoxa-4, hoxb-4, and hoxd-4. In contrast to the mutants of the paralogous family members, hoxc-4 homozygotes do not manifest abnormalities in the cervical vertebrae, but instead show vertebral defects that extend from the second thoracic vertebra (t2) to t11. Therefore, defects do not correspond to the anterior limit of expression of hoxc-4, but rather begin within the region of strong hoxc-4 expression in the prevertebral anlagen (i.e., pv7-14). While hoxc-4 mutant homozygotes that reach adulthood are fertile and appear outwardly normal, most die before weaning age. The high lethality appears to result from partial or complete blockage of the lumen of the esophagus over a large portion of its length, as well as disorganization of the esophageal musculature. Although the Drosophila homolog of hoxc-4, Deformed, is autoregulated, mutation of the hoxc-4 gene does not affect transcription of its paralogous family members. However, in hoxc-4 mutant embryos, transcription of both the hoxc-5 and hoxc-6 genes is altered. Employment of cissolidustrans analysis showed that the hoxc-4 mutation acts in cis to affect the pattern of hoxc-5 expression. Therefore, this mutation is likely to cause a reduction of hoxc-5 function as well as complete loss of hoxc-4 function.