AbstractWe established a two‐dimensional electrophoresis (2‐DE) mapping database of splenic CD4 T cells prepared from I‐Ad‐restricted ovalbumin (OVA)323‐339 specific T cell receptor (TCR) transgenic mice (OVA23‐3). First we examined the purification of CD4 T cells and found that the high purity of cells produced more accurate protein maps. The first dimension utilized narrow‐range immobilized pH gradients (IPGs), pH 4.0–5.0, pH 4.5–5.5, pH 5.0–6.0, and pH 5.5–6.7. Approximately 1300 spots were detected by silver staining. Detection was performed by in‐gel tryptic digestion of the spots, matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS) technology and database searches via the world wide web (WWW). We have so far identified 255 proteins on 2‐DE gels of whole cell lysates. This is the first construction of a proteome database for murine unsensitized CD4 T lymphocytes. To examine this further, 2‐DE mapping was utilized for splenic CD4 T cells from another TCR transgenic mouse strain (DO11.10 TCR transgenic mice). Mapping patterns were found to be almost identical to those from CD4 T cells from OVA23–3 mice. These results indicated that the 2‐DE maps in this study could be used for mouse CD4 T cells to examine protein changes in cells given certain stimuli.