The transient expression of estrogen receptor alpha (ERalpha) in the facial nucleus of rats during development was already reported. However, how and whether the receptor functions physiologically in the nucleus of developing rats are as yet unclear. In this study, we applied a retrograde tracer into one of the possible target muscles of the motoneurons in the nucleus, that is, the transverse auricular muscle (Mta), and examined whether ERalpha-immunopositive neurons take up the tracer. Because it is probable that neurogenesis, apoptosis, and maturation may be associated with the transient expression of ERalpha, we attempted to analyze the neurons expressing the receptor in the nucleus. We found that ERalpha-immunopositive neurons in the medial facial subnucleus innervate mostly the Mta. Quantitative analyses showed that the number of motoneurons projecting to the Mta remained the same throughout the ages examined, whereas that of ERalpha-immunopositive neurons decreased between postnatal days 6 and 11. Apoptosis and neurogenesis in the nucleus were not affected by the expression of ERalpha during development. ERalpha expression coincided with the maturation of neurons in the nucleus. Thus, it is possible that ERalpha expression in the facial nucleus during development plays important roles in the development of motoneurons and/or external pinna muscles.