Little is known regarding the functional effects of common autoimmune susceptibility variants on human immune cells. The SNP CT60 (rs3087243; A/G) located in the 3' UTR of the CTLA4 gene has been associated with autoimmune diseases. We examined a cohort of healthy individuals stratified by genotypes at CTLA4 to gain insight into the functional effects of allelic variation on T cell signaling. Using phospho-site-specific mAbs, we tested the hypothesis that the CT60 genotype at CTLA4 is associated with altered T cell antigen receptor (TCR) signaling in naive and/or memory T cells. By normalizing for the extent of the initial TCR signaling event at CD3zeta, we observed that the relative responsiveness to TCR stimulation as assessed by phosphorylation levels of downstream signaling molecules was altered in naive (CD4(+)CD45RA(high)) and memory (CD4(+)CD45RA(low)) T cells obtained from individuals with the disease-susceptibility allele at CTLA4. Thus, allelic variation associated with autoimmune disease can alter the signaling threshold of CD4(+) T cells. These experiments provide a rational approach for the dissection of T cell-susceptibility genes in autoimmune diseases.