We have recently shown that down-regulation of mouse Thy28 (mThy28) protein expression appears to be accompanied by apoptotic processes. Thymocytes from mice contain moderate amounts of mThy28 protein and undergo proliferation, differentiation, or apoptosis during murine thymic maturation. As a first step to examine the potential role of the mThy28 protein in the thymocyte development, such as positive-negative selection, the expression of mThy28 protein in the thymocyte subsets was examined. Thymocytes are separated into four subpopulations by the expression levels of CD4 and CD8: CD4-CD8- (DN), CD4+CD8+ (DP), and CD4+CD8- or CD4-CD8+ (SP). Flow cytometry analysis using three-color staining demonstrated that the mThy28 expression in immature DP cells is lower than that in DN and SP cells. The down-regulation of the mThy28 expression in the DP stage was also detected by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). The immunostaining method also showed that mThy28 protein was expressed in the medulla containing mature thymocytes, but not the cortex having immature thymocytes. The mThy28 protein in the thymocytes was mainly localized in the nucleus, as recently demonstrated in lymphoma cells, indicating that the mThy28 protein resides in the nucleus, irrespective of the cyclic or resting stage of the cell cycle. Together, the observation that mThy28 expression is down-modulated during the DP stage suggests that mThy28 protein might play some role in the positive-negative selection step in thymic maturation.