In most cases, cytotoxic T cell activation is dependent on the interaction of the T cell receptor (TCR) and CD8 with MHC class I molecules. In the CD8(+) T cell system based on the mouse cytotoxic T cell clone 2C, recognition of the allogeneic MHC L(d) exhibits a less significant role for CD8 than recognition of the syngeneic MHC K(b). Here, we examined whether this difference is related to the relative abilities of the two pepMHC complexes to drive the association of CD8 and TCR on the T cell surface. We show that both the syngeneic and allogeneic pepMHC induced association of CD8 and TCR, as revealed by fluorescence resonance energy transfer (FRET). Thus, the orientation of the syngeneic and allogeneic ligands when bound to the same TCR both allow CD8 to be recruited to the TCR complex. The conserved diagonal orientation of TCRs on different pepMHC ligands may facilitate such associations. The FRET results are consistent with the known binding properties and the CD8 involvement of the two different TCR:pepMHC interactions.