We established a two-dimensional electrophoresis (2-DE) mapping database of splenic CD4 T cells prepared from I-A(d)-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.