Engagement of MHC class II and CD40 on B cell lines triggers intracellular signals that activates cell surface adhesion receptors, resulting in LFA-1-dependent and -independent cell-cell adhesion. In this study, a murine monoclonal antibody (mAb R21) has been produced against a LFA-1-negative human B cell line and proven to completely block MHC class II- and CD40-induced LFA-1-independent homotypic adhesion. However, this mAb failed to prevent MHC class II- or CD40-induced homotypic adhesion in LFA-1-positive Raji B cells, and alone, it triggered LFA-1-dependent cell-cell adhesion. Biochemical characterization indicated that the CD20 molecule, a tetraspan phosphoprotein expressed on B cells that functions as a Ca2+-conductive ion channel, is the target of mAb R21. Interestingly, further biochemical analysis demonstrated that CD20 is physically associated with MHC class II and CD40 molecules on the cell surface of LFA-1-negative and LFA-1-positive B cell lines. Although these three molecules are associated with each other, the complex formation between any two of them is not dependent on the simultaneous expression of the three molecules. Altogether, these results indicate that CD20 is physically and probably functionally coupled to the MHC class II and CD40 molecules; thereby it may have certain modulatory effects on their functions.