Understanding the mechanisms of thymus regeneration is necessary for designing strategies to enhance host immunity when immune function is suppressed due to T cell depletion. In this study, expressed sequence tag (EST) analysis was performed following generation of a regenerating thymus cDNA library to identify genes expressed in thymus regeneration. A total of 1,000 ESTs were analyzed, of which 770 (77%) matched to known genes, 178 matched to unknown genes (17.8%) and 52 (5.2%) did not match any known sequences. The ESTs matched to known genes were grouped into eight functional categories: gene/protein synthesis (28%), metabolism (24%), cell signaling and communication (17%), cell structure and motility (6%), cell/organism defense and homeostasis (6%), cell division (3%), cell death/apoptosis (2%), and unclassified genes (14%). Based on the data of RT-PCR analysis, the expression of TLP, E2IG2, pincher, Paip2, TGF-β1, 4-1BB and laminin α3 genes was increased during thymus regeneration. These results provide extensive molecular information, for the first time, on thymus regeneration indicating that the regenerating thymus cDNA library may be a useful source for identifying various genes expressed during thymus regeneration.