CD45, a receptor-type tyrosine phosphatase, is required for interleukin-6 (IL-6)-induced proliferation in human myeloma cells, which express the shortest isoform, CD45RO, but not the longest isoform, CD45RA. Here, we showed that IL-6 induced the translocation of CD45 to lipid rafts in an isoform-dependent manner. In myeloma cells, CD45RO was translocated to lipid rafts more rapidly than CD45RB, but exogenously expressed CD45RA was not translocated. When an IL-6Ralpha-transfected B-cell line was stimulated with IL-6, CD45RA was not translocated, although CD45RB was. We further confirmed that the translocated CD45 bound to IL-6Ralpha, Lyn, and flotillin-2, and this was followed by the dephosphorylation of the negative regulatory Tyr507 of Lyn. CD45 also bound to phosphoprotein associated with glycosphingolipid-enriched microdomains (PAGs), which were subsequently dephosphorylated, resulting in the release of C-terminal src kinase (Csk) from lipid rafts. Therefore, these results indicate that a rapid translocation of CD45RO to lipid rafts may be responsible for IL-6-induced proliferation, and that the change from CD45RA to CD45RO confers the ability to respond to IL-6 in human myeloma cells.