T cell activation requires two signals: specific recognition of antigen through the T cell receptor (TCR) and a costimulatory signal provided primarily by CD28 in naïve T cells. We cloned a novel gene with considerable homology to RIBP/TSAd/Lad, an adaptor involved in T cell activation and interleukin-2 (IL-2) promoter activation. Expression of this gene is limited to the spleen and thymus. We have named this gene ALX, adaptor in lymphocytes of unknown function X. Because the related adaptor RIBP is involved in IL-2 regulation, we investigated whether ALX had a similar function. ALX overexpression in Jurkat T cells results in inhibition of IL-2 promoter activation after stimulation with superantigen. The IL-2 promoter contains several binding sites for transcription factors including the composite element RE/AP, which is the primary site of CD28 transcriptional activation. ALX overexpression had the greatest effect on the activation of a RE/AP reporter as opposed to an AP-1 reporter. Interestingly, ALX overexpression strongly inhibited RE/AP activation in response to anti-CD28/phorbol 12-myristate 13-acetate (PMA) stimulation but had minimal effect when anti-TCR/PMA was used. Therefore, it appears that ALX may function downstream of CD28 costimulation during T cell activation. In addition, the mobility of ALX shifts upon TCR/CD28 costimulation to a greater extent than what is observed with either stimulus alone demonstrating that ALX is a target of both TCR and CD28 costimulatory signaling pathways.