In this study experiments were conducted to elucidate the physical/functional relationship between CD45 and casein kinase 2 (CK2). Immunoprecipitation experiments demonstrated that CK2 associates with CD45 and that this interaction is inducible upon Ag receptor cross-linking in B and T cell lines as well as murine thymocytes and splenic B cells. However, yeast two-hybrid analysis failed to demonstrate a physical interaction between the individual CK2 α, α′, or β subunits and CD45. In contrast, a yeast three-hybrid assay in which either CK2 α and β or α′ and β subunits were coexpressed with the cytoplasmic domain of CD45, demonstrated that both CK2 subunits are necessary for the interaction with CD45. Experiments using the yeast three-hybrid assay also revealed that a 19-aa acidic insert in domain II of CD45 mediates the physical interaction between CK2 and CD45. Structure/function experiments in which wild-type or mutant CD45RA and CD45RO isoforms were expressed in CD45-deficient Jurkat cells revealed that the 19-aa insert is important for optimal CD45 function. The ability of both CD45RA and CD45RO to reconstitute CD3-mediated signaling based on measurement of calcium mobilization and mitogen-activated protein kinase activation was significantly decreased by deletion of the 19-aa insert. Mutation of four serine residues within the 19-a insert to alanine affected CD45 function to a similar extent compared with that of the deletion mutants. These findings support the hypothesis that a physical interaction between the CD45 cytoplasmic domain and CK2 is important for post-translational modification of CD45, which, in turn, regulates its catalytic function.