Cloned T cell lines specific for the antigen ovalbumin (OVA) in the context of self I-A or I-E class II MHC-encoded molecules were found to express equivalent levels of the Lyt-2 and L3T4 surface molecules by FACS analysis. Functionally, these cloned T cell lines will kill OVA-pulsed, class II MHC-bearing B lymphoma cells. This system allowed us to examine the relative contribution of the Lyt-2 and L3T4 molecules to recognition of antigen in the context of self class II MHC molecules. We find that anti-L3T4 is 25- to 100-fold more potent at inhibiting cytotoxicity by these cloned lines than is anti-Lyt-2, where potency is calculated as the ratio of the concentration needed for inhibition divided by the concentration needed for binding. Both antibodies can completely inhibit cytotoxicity. These results suggest that the accessory molecules Lyt-2 and L3T4 can play two different roles in antigen:MHC recognition. In class II MHC recognition, L3T4 plays a dominant role, highly sensitive to inhibition by anti-L3T4. By contrast, Lyt-2 plays a minor yet important role in the cell interaction, perhaps by adhesion strengthening. Previous analysis in several laboratories supports the concept that the ligand for L3T4 is class II MHC, whereas the ligand for Lyt-2 is class I MHC. The present results are consistent with the hypothesis that these molecules are actually part of the T cell antigen:MHC recognition complex, and play an important role in the class of MHC molecule recognized by a T cell alpha:beta heterodimeric receptor. As such, they are not accessory proteins, but a direct part of the recognition apparatus, and the term accessory protein may apply only in those cases in which the specificity of the T cell receptor is for a different class of MHC than that normally associated with the L3T4 or Lyt-2 molecule.