The receptor-like protein tyrosine phosphatase CD45 is essential for TCR signal transduction. Substrates of CD45 include the protein tyrosine kinases p56 lck and p59 fyn, both of which have been shown to be enriched in detergent-insoluble microdomains. Here we find that there is a cholesterol-dependent association between CD45 and the raft-associated protein linker for activation of T cells, suggesting that CD45 and linker for activation of T cells may colocalize in lipid rafts. Consistent with this observation, we find that∼ 5% of total CD45 can be detected in Triton X-100-insoluble buoyant fractions of sucrose gradients, demonstrating that CD45 is not excluded from lipid rafts. Upon stimulation of T cells with anti-CD3, there is a reduction in the amount of CD45 found associating with lipid rafts. Our data suggest that CD45 is present in lipid rafts in T cells before activation, perhaps to activate raft-associated p56 lck, allowing membrane-proximal signaling events to proceed. Furthermore, the reduction in CD45 content of lipid rafts after CD3 stimulation may serve to limit the amounts of activated p56 lck in rafts and thus possibly the duration of T cell responses.