The ephrin/Eph system is well known to regulate various aspects of brain development. In this study, we analyzed the expression profiles of EphA3 at both the RNA and protein level in developing mouse forebrains. Although the EphA3 gene is known to encode two isoforms of the receptors, a full-length transmembrane form, and a short, secretory form, only the full-length isoform was detected in the developing forebrain. We found that, in the early developmental stages, while EphA3 mRNA was expressed in the dorsal thalamus and the cortical intermediate zone (IMZ), the EphA3 protein was detected in the IMZ and the internal capsule, but not in the dorsal thalamus. In the later stages the mRNA was expressed in the most superficial region of the cortical plate, while the protein was expressed in the IMZ. This discrepancy between the mRNA and protein expression patterns might be attributed to the possibility of the protein being transported to the axons to regulate the thalamocortical and corticofugal projection. The results of double-immunostaining for L1 and EphA3 or TAG-1 and EphA3 suggested that EphA3 protein was produced mainly in the thalamocortical axons and only partially in the corticofugal axons. In addition, the EphA3 protein was also detected in various other structures, such as the lateral olfactory tract, anterior commissure, and corpus callosum, suggesting the possibility that EphA3 might regulate the formation of various neuronal networks in the developing brain, including the TC projection and the commissural fibers. J. Comp. Neurol. 487:255–269, 2005. © 2005 Wiley-Liss, Inc.