The embryonic events surrounding tracheo-esophageal separation remain controversial. The present study was undertaken to clarify early tracheo-bronchial development in the rat embryo at a critical period of organogenesis. Twenty-seven timed-mated Sprague-Dawley rats were divided into nine groups of three rats. Their embryos were harvested on gestational days 11-15 at intervals of 8 h, processed and sectioned transversely. The sections were stained with hematoxylin and eosin and examined serially. The foregut is a single tube on gestational day 11. During the following 16 h, there is localized and rapid growth of the respiratory epithelium and a laterocaudal expansion to form the bronchial buds and a protuberance on the ventral wall of the foregut (future tracheal carina). From gestational days 12-12 + 8, cellular debris and apoptotic epithelial cells are specifically located in the tracheo-esophageal groove, resulting in collapse and fusion of the lateral walls of the foregut, effectively separating the trachea and esophagus. Afterwards, the epithelial proliferation dominates the process of tracheo-esophageal separation until it reaches the caudal end of the laryngeal epithelial lamina on gestational day 15. The present study shows that separation of the trachea from the esophagus involves three consecutive stages: (i) epithelial proliferation resulting in the formation of bronchial buds and the tracheal carina; (ii) epithelial apoptosis leading to separation of the trachea and esophagus; and (iii) epithelial proliferation to complete the separation process.