Studies of chick-quail chimeras have reported that avian ultimobranchial C cells originate from the neural crest. It has consequently been assumed, without much supporting evidence, that mammalian thyroid C cells also originate from the neural crest. To test this notion, we employed both Connexin43-lacZ and Wnt1-Cre/R26R transgenic mice, because their neural crest cells can be marked. We also examined the immunohistochemical expression of a number of markers that identify migratory or postmigratory neural crest cells, namely, TuJ1, neurofilament 160, nestin, P75NTR, and Sox10. Moreover, we examined the expression of E-cadherin, an epithelial cell marker. At embryonic day (E)10.5, the neural crest cells densely populated the pharyngeal arches but were not distributed in the pharyngeal pouches, including the fourth pouch. At E11.5, the ultimobranchial rudiment formed from the fourth pouch and was located close to the fourth arch artery. At E13.0, this organ came into contact with the thyroid lobe, and at E13.5, it fused with this lobe. However, the ultimobranchial body was not colonized by neural crest-derived cells at any of these developmental stages. Instead, all ultimobranchial cells, as well as the epithelium of the fourth pharyngeal pouch, were intensely immunoreactive for E-cadherin. Furthermore, confocal microscopy of newborn mouse thyroid glands revealed colocalization of calcitonin and E-cadherin in the C cells. The cells, however, were not marked in the Wnt-Cre/R26R mice. These results indicated that murine thyroid C cells are derived from the endodermal epithelial cells of the fourth pharyngeal pouch and do not originate from neural crest cells.