CD45, the major transmembrane tyrosine phosphatase of lymphoid cells, is required for optimal signaling via a number of receptors. A model for how CD45 regulates signaling is that it controls phosphorylation of the COOH-terminal tyrosine of src family kinases. We have shown that CD45 physically associates with lck, one src kinase. Others have shown that CD45 also interacts with the CD4 and CDB surface antigens expressed on many T cells. In this report we examine further the relationship between CD45 and lck in a CD4+ T cell line and in peripheral T cells. We show now that CD45 associates with lck independently of both CD4 and CD8. We show also the time course of an association between CD45 and a form of lck that migrates at an apparent higher molecular mass. Finally, we demonstrate that the interaction between CD45, lck, and a previously reported 32-34 kD protein is stable after stimulation of T cells. © 1994 Academic Press, Inc.