Members of the NK family of homeobox transcription factors regulate critical steps of organogenesis during vertebrate development. In the studies described in this report, we have isolated and functionally characterized the chickenNkx-2.8(cNkx-2.8) cDNA and protein and defined the temporal and spatial pattern ofcNkx-2.8gene expression during chicken development.cNkx-2.8transcripts are first detectable at HH stage 7 in the splanchnopleura. At stage 10+, thecNkx-2.8gene is expressed in the linear heart tube and the dorsal half of the vitelline vein. However, after looping, HH stage 13,cNkx-2.8is no longer expressed in the looped heart tube, but is expressed in the ventral pharyngeal endoderm. At stage 15, in addition to the pharyngeal expression pattern,cNkx-2.8is expressed in the ectoderm of the pharyngeal arches and the aortic sac. By HH Stage 17,cNkx-2.8expression is detectable in lateral endoderm of the second and third pharyngeal pouches, the posterior portion of the aortic sac, and the sinus venosus. cNkx-2.8 binds to previously characterized Nkx2-1 and Nkx2-5 DNA-binding sites and overexpression of cNkx-2.8 transactivates a minimal promoter which contains multimerized Nkx-2 DNA-binding sites. In addition, cNkx-2.8 and serum response factor can coactivate a minimal cardiac α-actin promoter. These data are consistent with a model in which cNkx-2.8 performs a unique temporally and spatially restricted function in the developing embryonic heart and pharyngeal region. Moreover, the coexpression ofcNkx-2.5and-2.8raises the possibility that cNkx-2.8 may have a redundant role with cNkx-2.5 in the coalescing heart tube and may play an important role in the transcriptional program(s) that underlies thymus formation. The existence of multiple NK-2 family members and their partially overlapping patterns of expression are discussed within the framework of a “Nkx code.”