B6.H-2Kb-/-Db-/- (DKO) mice have greatly reduced numbers of mature CD8αβ T cells in their periphery. However, these non-class Ia-selected CD8αβ T cells are able to mediate immune responses to a number of pathogens. Approximately 60% of the CD8αβ T cells in the spleen and peripheral lymph nodes of naive DKO mice display a memory (CD44 high) phenotype. To investigate the origins of these non-class Ia-selected CD8αβCD44high cells, we traced the phenotype of recent thymic emigrants and found that most were CD44low. We also determined whether their appearance was thymus dependent and found that only a small percentage of non-class Ia-selected CD8αβCD44high cells develop in a thymus-independent pathway. Functionally, CD8αβCD44high cells from DKO mice are able to secrete IFN-γ in response to IL-12 and IL-18 in the absence of cognate Ag. When challenged with anti-CD3 in vivo, nearly half of these cells produce IFN-γ within 3 h. When panned CD8αβCD44high cells from Thy1.2.DKO mice were transferred into Thy1.1 DKO recipients and then challenged with Listeria monocytogenes, an Ag-specific anti-L. monocytogenes response was observed 6 days later. Our data suggest that non-class Ia-selected CD8αβCD44high cells in naive animals can respond rapidly to Ag and play a role in the innate as well as the early phase of the acquired immune response.