Lipopolysaccharide (LPS) from gram-negative bacteria causes polyclonal activation of B cells and stimulation of macrophages and other APC. We show here that, under in vivo conditions, LPS also induces strong stimulation of T cells. As manifested by CD69 upregulation, LPS injection stimulates both CD4 and CD8⁺ T cells, and, at high doses, stimulates naive (CD44^lo) cells as well as memory (CD44^hi) cells. However, in terms of cell division, the response of T cells after LPS injection is limited to the CD44^hi subset of CD8⁺ cells. In contrast with B cells, proliferative responses of CD44^hi CD8⁺ cells require only very low doses of LPS (10 ng). Based on studies with LPS-nonresponder and gene-knockout mice, LPS-induced proliferation of CD44^hi CD8⁺ cells appears to operate via an indirect pathway involving LPS stimulation of APC and release of type I (α, β) interferon (IFN-I). Similar selective stimulation of CD44^hi CD8⁺ cells occurs in viral infections and after injection of IFN-I, implying a common mechanism. Hence, intermittent exposure to pathogens (gram-negative bacteria and viruses) could contribute to the high background proliferation of memory–phenotype CD8⁺ cells found in normal animals.