Although it is clear that the CD45 tyrosine phosphatase is required for efficient T‐cell activation and T‐cell development, the factors that regulate CD45 function remain uncertain. Previous data have indicated that there is an association of CD45 with CD4 and the T‐cell receptor (TCR) complex controlled by the variable ectodomain of CD45 and, following activation, by high‐ and low‐potency peptides. This suggests that controlling substrate access to CD45 may be an important regulatory mechanism during T‐cell activation. In the present study we have examined the role of the transmembrane adapter‐like molecule CD45‐associated protein (CD45‐AP) in regulating the association of CD45 with CD3/TCR and lck, and in regulating primary CD4⁺ T‐lymphocyte activation. In CD4⁺ T cells from CD45‐AP‐deficient mice, coimmunoprecipitation of CD45 with the CD3/TCR complex, in addition to lck, is significantly reduced compared with wild‐type T cells. Functionally, this correlates with a decreased proliferative response, a decrease in interleukin (IL)‐2 production, and a decrease in calcium flux upon stimulation with a low‐potency altered peptide ligand. However, the response of CD45‐AP‐deficient T cells to stimulation with a high‐avidity agonist peptide was largely intact, except for a modest decrease in IL‐2 production. These data suggest that CD45‐AP promotes or stabilizes the association of CD45 with substrates and regulates the threshold of T‐cell activation.