Activation of human PBL T cells with solid phase anti-CD3 mAb or during the course of an MLR response gives rise to the association of CD4 or CD8 molecules with the protein tyrosine phosphatase, CD45, on the cell surface. This paired association of cell-surface molecules occurs late in the activation cycle and appears to be dependent upon Ti-CD3-mediated signaling because mitogen-driven activation does not induce formation of the complex. Maximal association occurred 72 to 96 h after exposure to anti-CD3 mAb on both CD4+ and CD8+ T cells. In contrast, association between CD8 and CD45 during an MLR response did not occur until day 6 of a MLR whereas CD4-CD45 association was detected by 72 h of culture. The kinetics of association between CD4 or CD8 and CD45 was measured by fluorescence resonance energy transfer and confirmed by immunoprecipitation of dithiobis succinimidylpropionate or disuccinimidyl suberate cross-linked 125I-labeled resting or activated T cells. The molecules that co-precipitated with either CD4 or CD8 and had an apparent kDa of 180 to 205 could be immunodepleted with anti-CD45 mAb. Furthermore, CD4 or CD8 immunoprecipitates from 96-h activated T cells contained significant levels of protein tyrosine phosphatase activity whereas corresponding immunoprecipitates from resting or recently activated T cells showed little protein tyrosine phosphatase activity. This association may allow CD45 to engage and dephosphorylate lck or another CD4- or CD8-associated substrate in order to reset the receptor complex to receive a new set of stimuli. Our observations suggest that synergistic signaling provided as a consequence of CD4 or CD8 association with the TCR after antigenic stimulation may develop on a different temporal scale than that observed after soluble anti-CD4+ anti-CD3 heteroconjugate antibody cross-linking.