The mechanism of CD8 cooperation with the TCR in antigen recognition was studied on live T cells. Fluorescence correlation measurements yielded evidence of the presence of two TCR and CD8 subpopulations with different lateral diffusion rate consts. Independently, evidence for two subpopulations was derived from the exptl. obsd. two distinct assocn. phases of cognate peptide bound to class I MHC (pMHC) tetramers and the T cells. The fast phase rate const. ((1.7) * 105 M-1 s-1) was independent of examd. cell type or MHC-bound peptides' structure. Its value was much faster than that of the assocn. of sol. pMHC and TCR ((7.0) * 103 M-1 s-1), and close to that of the assocn. of sol. pMHC with CD8 ((1-2) * 105 M-1 s-1). The fast binding phase disappeared when CD8-pMHC interaction was blocked by a CD8-specific mAb. The latter rate const. was slowed down .apprx. 10-fold after cells treatment with methyl-b-cyclodextrin. These results suggest that the most efficient pMHC-cell assocn. route corresponds to a fast tetramer binding to a colocalized CD8-TCR subpopulation, which apparently resides within membrane rafts: the reaction starts by pMHC assocn. with the CD8. This markedly faster step significantly increases the probability of pMHC-TCR encounters and thereby promotes pMHC assocn. with CD8-proximal TCR. The slow binding phase is assigned to pMHC assocn. with a noncolocalized CD8-TCR subpopulation. Taken together with results of cytotoxicity assays, the authors' data suggest that the colocalized, raft-assocd. CD8-TCR subpopulation is the one capable of inducing T-cell activation. [on SciFinder (R)]