The adaptor protein ALX acts downstream of CD28 to regulate the interleukin-2 (IL-2) promoter during T cell activation. Whereas ALX is predominantly localized to the cytoplasm, ALX partially resides in the nucleus, and the nuclear pool is rapidly depleted in response to T cell receptor (TCR)/CD28 signaling. Here it is shown that this depletion occurs via nuclear export of ALX, which depends on a leucine-rich nuclear export signal (NES) in its carboxyl segment and on the CRM-1 transport protein. Nuclear import of ALX also depends on its carboxyl-terminal segment. Blocking nuclear export of ALX, either pharmacologically, by leptomycin B, or by site-directed mutation of the ALX NES, impairs CD28-mediated phosphorylation of ALX. Additionally, upon overexpression, the ALX NES mutant was found to be impaired in inhibiting TCR/CD28-induced transcriptional up-regulation of the RE/AP composite element from the IL-2 promoter, whereas a truncated form of ALX that is a potent inhibitor of RE/AP activation was found to reside entirely in the cytoplasm. Together, these results show that ALX exerts its effect on IL-2 up-regulation in the cytoplasm and suggest an intricate relationship between the nuclear localization/export, phosphorylation, and activity of ALX in response to TCR and CD28 signaling.