Mice with a targeted deletion of the Hoxa3 gene have defects of derivatives of the third branchial arch and pouch. To address the role of the Hoxa3 gene in parathyroid organogenesis, we examined the third pharyngeal pouch development by immunohistochemistry (IHC) using the secretory protein (SP)-1/chromogranin A antiserum, which recognizes the parathyroid from its initial formation onward. At embryonic day (E) 11.5, the SP-1/chromogranin A-immunoreactive primary rudiment of the parathyroid appeared in the cranial region of the third pharyngeal pouch of wild-type embryos. In Hoxa3-null mutants, the third pharyngeal pouch was normally formed but failed to differentiate into the parathyroid rudiment, showing no immmunoreactivity for SP-1/chromogranin A. Classic studies using chick-quail chimeras have demonstrated that the ectomesenchymal neural crest cells are required for proper development of the pharyngeal pouch-derived organs, including the thymus and parathyroid glands. To visualize the migration and development of mesenchymal neural crest cells in Hoxa3 mutants, the heterozygotes were crossed with connexin43–lacZ transgenic mice in which β-galactosidase expression was specific to the neural crest cells. In Hoxa3 homozygotes and in wild types, ectomesenchymal neural crest cells densely populated the pharyngeal arches, including the third one, and surrounded the third pouch epithelium. These results indicate that lack of the Hoxa3 gene affects the intrinsic ability of the third pharyngeal pouch to form the parathyroid rudiment and has no detectable effect on the migration of neural crest cells.