Teeth are formed by reciprocal interactions between the epithelium and mesenchyme in the first pharyngeal arch. Although the contribution of midbrain and hindbrain crest cells to the first pharyngeal arch has been previously examined in rodent embryos, no direct evidence exists that these cells are actually involved in the dental mesenchyme. In order to elucidate the contribution of the cranial neural crest cells in tooth formation, we first identified the emigration sites and stages providing the crest cells that migrate to the presumed tooth-forming region of the mandibular prominence. Focal labeling with DiI was performed at the midbrain and anterior hindbrain crests in rat embryos, and the labeled embryos were cultured for 30 or 60 hr. The resultant migration patterns indicated that posterior midbrain crest cells emigrating by the end of the 4-somite stage predominantly migrated to the region where tooth buds normally develop. Second, we established a new type of long-term culture system in which whole embryo culture is followed by a mandibular organ culture. Using this system, rat embryos were maintained from the early-somite stage and the molars in the explants were able to reach the bud stage within 8 days. Finally, to ascertain if posterior midbrain crest cells emigrating by the end of the 4-somite stage were involved in the dental mesenchyme, these cells were labeled with DiI and processed for the long-term culture. Labeled crest cells were clearly detectable in the dental mesenchyme. These findings indicate that the early-emigrating posterior midbrain crest cells contribute to mandibular molar tooth development in rat embryos.