An isolated tracheo-oesophageal fistula could be caused by close proximity of the epithelia of both organs (O'Rahilly & Müller, 1984; Kluth et al. 1987) at certain embryonic stages, the most frequent location being the tracheal bifurcation. Thus the relative position and degree of separation between the digestive and the respiratory tubes throughout their development may be relevant to the origin of this anomaly. The aim of this study was to analyse along the different segments of the tracheo-oesophageal septum (TES) where the closest relationship between both lumina occurred and what degree of separation was present at each segment. Computer imaging techniques were applied on cross sections of a graded series of normal human embryos (Carnegie stages (CS) 13–23). In addition, the differentiation of the primitive TES was also studied (from CS 12) by light microscopy. Between CS 13 and 16 both tubes tended to separate (phase of separation), principally at the proximal segments of the laryngopharyngeal and the tracheo-oesophageal portions of the TES. During this phase the separation between the trachea and oesophagus was wider than between the larynx and pharynx. From CS 17 to CS 23 the digestive and respiratory lumina reached their widest separation at different levels of the laryngopharyngeal portion. Below these levels they tended to come closer together, principally at the proximal segment of the tracheo-oesophageal portion, but also at the distal part of the laryngopharyngeal portion. During this phase of approximation they reached their closest relationship at the proximal (CS 17) and the distal (from CS 18) segments of the tracheo-oesophageal portion. When finally the distal segment of the trachea (which includes the bifurcation) comes closest to the oesophagus, the coats of both organs have already undergone an appreciable differentiation. According to these observations, the origin of the most frequent isolated tracheo-oesophageal fistula at the bifurcation region could not be explained from the normal development of the TES.