The highly polymorphic human major histocompatibility complex (HLA) class II molecules are acknowledged as signaling receptors although their coupling to signaling pathways is not yet fully elucidated. In this study, we investigated how HLA class II can be coupled to protein tyrosine kinase (PTK) signaling pathway in B cells and whether there might be differences depending on HLA class II isotype. Using the human B cell line Ramos, we demonstrate that CD19 and CD20 are two HLA class II-associated receptors that couple HLA class II to PTK signaling pathway where CD20 appears to be amajor component of HLA class II-mediated activation of Src kinases. Both HLA-DR and HLA-DP co-immunoprecipitate tyrosine-phosphorylated proteins (p-Tyr) whereas only activation through HLA-DR increases the tyrosine phosphorylation of these proteins. Indeed, in contrast to HLA-DR, cross-linking HLA-DP induces neither tyrosine phosphorylation nor homotypic adhesion, and induces ERK1/2 activation. Differential association of these isotypes with CD20 appears to be one of the mechanisms underlying their differential signaling. We provide an experimental evidence for a mechanism by which HLA class II molecules can be coupled to PTK signaling pathway and, underscores their isotypes differential signaling. Further investigation of these mechanisms is likely to provide new insights into how isotype specific MHC class II signaling can contribute to the regulation of the immune response.