CD20 is a B cell surface protein which can initiate intracellular signals involving tyrosine kinase activation, and modify B cell growth and differentiation. CD20 is tightly associated with the Src-family kinases Lyn, Fyn and Lck; however, the mechanism of interaction remains to be determined. Association between CD20 and Src-family kinases has been detected in peripheral blood B cells and in 5 out of 8 unrelated B cell lines. The lack of CD20-associated kinase activity in some cell lines offered an opportunity to investigate the mechanism of CD20 associations. All 8 B cell lines were found to express Lyn, and, with one exception, all cell lines also expressed Fyn. Lck, however, was not detected in any of the cell lines in which CD20 failed to coprecipitate kinase activity. To test the possibility that Lck was required for assembly of the CD20 complex, Lck was transfected into one of the 3 CD20/kinase association-deficient lines, namely T51. CD20 did not coprecipitate kinase activity from the transfected T51 cells, despite their expression of high levels of exogenous Lck, as well as endogenous Lyn and Fyn. CD20 cDNA from T51 was sequenced and found to be normal. These data establish that association between CD20 and Src-family kinases requires an additional factor.