Background/Purpose: The embryogenesis of many congenital tracheoesophageal abnormalities has been elucidated poorly, mainly because of an incomplete understanding of the normal mechanism of separation of the primitive foregut into the trachea and esophagus. There has been controversy about the existence and significance of the so-called tracheoesophageal septum. This study investigates the normal mechanism of tracheoesophageal separation in the rat.Methods: Timed-pregnant Sprague-Dawley rats were killed on gestational days 11 to 16, respectively (the day on which the vaginal smear showed sperm was considered day 0 of gestation). Thirty-six embryos (six embryos from each age group) were fixed in 10% formalin. Fixed embryos from gestational day 13, 12, and 11 were sandwiched in melted agar to facilitate proper orientation before being processed. Half of the fetuses from each age group were sectioned serially in a transverse plane and the other half in a sagittal plane. The histological sections were stained with H&E.Results: On gestational day 11, the foregut appears as a ventrodorsal slit in transverse section. Below the primitive pharynx, the epithelial cells on the ventral part of the foregut are actively proliferating, producing two lateral epithelial bulges (primitive bronchial buds). The epithelial cells of the dorsal aspect of the foregut show no sign of active growth. On day 12, the proximal trachea still shares a common lumen with the foregut. The distal trachea, the tracheal bifurcation, and both primary bronchi are recognized easily. At the point of tracheoesophageal separation, there is obvious debris of dead epithelial cells and condensed nuclei. These appearances were specifically located around the so-called tracheoesophageal septum and nearby grooves, resulting in enfolding cristae. There was no cell proliferation or inflammatory response in this region. On gestational day 13, the separation of the trachea and esophagus was almost complete.Conclusion: The present study demonstrates that a specific and consistent pattern of cell death in the region of the ''tracheoesophageal septum'' produced enfolding cristae that may be part of the mechanism of separation of the trachea and esophagus.