We found that the number of T cell receptor (TCR)αβ+ CD4− CD8− T cells increased in the peritoneal cavity on day 5 after an intraperitoneal infection with Listeria monocytogenes strain EGD together with TCRγδ+ CD4− CD8− T cells. Thereafter, the TCRαβ+ CD4− CD8− T cells decreased to a normal level by day 14. The TCRαβ+ CD4− CD8− T cells showed an activated T cell phenotype (L-selectin-CD44+) and expressed CD45/B220 and interleukin-2 receptor β, but did not express heat stable antigen, which is expressed by the immature CD4− CD8− thymocytes. Furthermore, 20–30% of the TCRαβ+ CD4− CD8− T cells expressed the NK1.1 natural killer cell marker. Analyses of the TCR V region repertoire of the TCRαβ+ CD4− CD8− T cells induced by L. monocytogenes infection showed that more than 80% of the TCRαβ+ CD4− CD8− T cells expressed TCR Vβ8 detected by anti-TCR Vβ8.1 and 8.2 mAb, and a reverse transcription-polymerase chain reaction analysis of Vα14 relative to Vα11 expression revealed that the TCRαβ+ CD4− CD8− T cells expressed a higher level of Vα14, which was reported to be preferentially expressed by TCRαβ+ CD4− CD8− thymocytes rather than conventional CD4+ T cells. The TCRαβ+ CD4− CD8− T cells showed a proliferative response to anti-TCRαβ mAb stimulation. In contrast, they showed no response to stimulation with either Listeria antigen or 65-kDa heat shock protein of Mycobacterium bovis, which do stimulate the Listeria-specific TCRαβ+ CD4+ CD8− T cells and the Listeria-induced TCR γδ+ T cells, respectively. These results suggest that the TCRαβ+ CD4− CD8− T cells may recognize a restricted set of self antigens induced by L. monocytogenes infection, and that they contribute to host protection at an early stage of the infection.