Cell-surface microvilli play a central role in adhesion, fusion, and signaling processes. Some adhesion and signaling receptors segregate on microvilli but the determinants of this localization remain mostly unknown. In this study, we considered CD4, a receptor involved in immune response and HIV infection, and p56(Lck), a CD4-associated tyrosine kinase. Analysis of CD4 trafficking reveals that p56(Lck) binds tightly to CD4 independently of its activation state and inhibits CD4 internalization. Electron microscopy analysis established that p56(Lck) mediates CD4 association with microvilli whereas biochemical data indicate that p56(Lck) expression renders CD4 insoluble by the nonionic detergent Triton X-100. In addition, cytoskeleton-disrupting agent increased CD4 solubility, suggesting the involvement of cytoskeletal elements in CD4 anchoring to microvilli. This concept was supported further by the observation that the lateral mobility of CD4 within the plasma membrane was decreased in cells expressing p56(Lck). Finally, isolation of detergent-resistant membranes revealed that the complex CD4-p56(Lck) is enriched within these domains as opposed to conditions in which CD4 does not interact with p56(Lck). In conclusion, our results show that p56(Lck) targets CD4 to specialized lipid microdomains preferentially localized on microvilli. This localization, which prevents CD4 internalization, might facilitate CD4-mediated adhesion processes and could correspond to the signaling site of the receptor.