Ly-6C has been proposed as a marker of memory CD8+ T cells. Reports have indicated that Ly-6C is upregulated after T cell receptor (TCR) stimulation or exposure to proinflammatory cytokines. This study examined the relative roles of proinflammatory cytokines and TCR engagement in Ly-6C induction. In vitro experiments tested the effects of cytokines on Ly-6C expression and confirmed interferon-alpha (IFN-alpha) as a primary cytokine that induces Ly-6C expression on CD4+ and CD8+ T cells. The amount and duration of Ly-6C expression were examined on T cells after in vivo induction of proinflammatory cytokines (CpG oligodeoxynucleotides [ODN]) or TCR activation (staphylococcal enterotoxin B [SEB]). In vivo, proinflammatory cytokines transiently upregulated Ly-6C on T cells in the absence of TCR stimulation. TCR stimulation by SEB transiently upregulated Ly-6C expression on antigen-specific and antigen-nonspecific T cells but did not cause long-term upregulation of Ly-6C expression in either population. IFN-alpha was confirmed as a primary inducer of Ly-6C in vivo, as CpG ODN were unable to induce Ly-6C expression in IFN-alphaRI(-/-) mice. Thus, inducible Ly-6C expression on CD4+ and CD8+ T cells is largely due to IFN-alpha in the environment and appears not to be directly correlated with the development of T cell memory.