Programmed cell death (PCD) is an essential event for development. The purpose of this work was to ascertain how PCD, in vivo designated apoptosis, is involved in the development of the external auditory canal. We performed a time sequence study of the distribution of apoptosis during the development of external auditory canal (EAC) of the mouse. ICR mice ranging in age from embryonic day 11.5 (E11.5) to 12 days after birth (DAB) were used in the present study. A part of each head including both ears was removed and was processed according to its purpose. Light and electron microscopy for morphological studies and TUNEL method (Gavrieli et al. [1992] J Cell Biol., 119:493-501) for histochemical studies were used. On E11.5, distinct TUNEL-positive staining occurred in the branchial arch. Between E15.5 and 1DAB, TUNEL-positive cells were observed throughout the EAC and the number of these cells decreased with age. On E15.5 and E16.5, numerous TUNEL-positive cells were observed in a cavity remained in the epithelial plate. Transmission electron microscopy revealed that these cells had the features of apoptosis. From 3-12 DAB, no apoptosis was observed in the EAC except for the terminal differentiation of the skin of the EAC. Apoptosis was not observed during recanalization of the EAC, but occurred during the formation of the epithelial plate. The investigation established that PCD is involved in the formation of the epithelial plate, whereas only cornification of the epithelium of the EAC is associated with recanalization.