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An investigation into the role of CD45 isoforms in T cell antigen receptor signal transduction was carried out by transfecting CD45-negative CD4+CD8+HPB-ALL T cells with the CD45R0, CD45RBC, and CD45RABC isoforms. Fluorescence resonance energy transfer analysis showed that the CD45R0 isoform, but not the CD45RBC or CD45RABC isoforms, was found as homodimers and also preferentially associated with CD4 and CD8 at the cell-surface. A comparison was therefore made of T cell antigen receptor signaling between sub-clones expressing either CD45R0 or CD45RBC. Under basal conditions CD4-associated p56lcktyrosine kinase activity and cellular protein tyrosine phosphorylation levels were higher in the CD45R0+than in the CD45RBC+sub-clones. Upon CD3-CD4 ligation, TCR-ζ phosphorylation, ZAP-70 recruitment to the p21/p23 TCR-ζ phosphoisomers, ZAP-70 phosphorylation, as well as p56lck, c-Cbl and Slp-76 phosphorylation, were all markedly increased in CD45R0+compared with CD45RBC+cells. T cell antigen receptor (TCR) stimulation alone also promoted c-Cbl phosphorylation in CD45R0+but not in CD45RBC+cells. Our results are consistent with a model in which association of CD45R0 with CD4 generates a more active pool of CD4-associated p56lckkinase molecules. Upon CD3-CD4 co-ligation, the active p56lckincreases the intensity of T cell antigen receptor signal transduction coupling by promoting TCR-ζ chain phosphorylation and ZAP-70 recruitment.