Hoxgenes encode transcription factors that are used to regionalize the mammalian embryo. Analysis of mice carrying targeted mutations in individual and multipleHoxgenes is beginning to reveal a complex network of interactions among these closely related genes which is responsible for directing the formation of spatially restricted tissues and structures. In this report we present an analysis of the genetic interactions between all members of the third paralogous group,Hoxa3, Hoxb3,andHoxd3.Previous analysis has shown that although mice homozygous for loss-of-function mutations in eitherHoxa3orHoxd3have no defects in common, mice mutant for both genes demonstrate that these two genes strongly interact in a dosage-dependent manner. To complete the analysis of this paralogous gene family, mice with a targeted disruption of theHoxb3gene were generated. Homozygous mutants have minor defects at low penetrance in the formation of both the cervical vertebrae and the IXth cranial nerve. Analysis and comparison of all double-mutant combinations demonstrate that all three members of this paralogous group interact synergistically to affect the development of both neuronal and mesenchymal neural crest-derived structures, as well as somitic mesoderm-derived structures. Surprisingly, with respect to the formation of the cervical vertebrae, mice doubly mutant forHoxa3andHoxd3orHoxb3andHoxd3show an indistinguishable defect, loss of the entire atlas. This suggests that the identity of the specificHoxgenes that are functional in a given region may not be as critical as the total number ofHoxgenes operating in that region.