(B6 X A)F1 mice were immunized with sperm whale myoglobin, and T cell clones and hybridomas were generated. Hybridoma 74a.e9 was specific for the sperm whale myoglobin 67-79 peptide and could be partially activated by a peptide analogue, equine myoglobin with a natural 74G substitution. Using this hybridoma in T cell activation assays, we studied the effects of varying the avidity of the TCR for its ligand, the concentration of MHC:peptide complex on the APC, and the density of TCR on the surface. Varying ligand concentration on the surface of the APC, the TCR avidity, or the density of TCR on the T cell were equally important parameters in driving T cell activation. The mouse myoglobin (74T) analogue, however, acted as an antagonist to the T cell response. Its effectiveness was also partially determined by its ability to bind to MHC. By independently altering each of these variables and following T cell activation, we describe the interrelationships among these three components (MHC:peptide:TCR) that control the activation of the T cell.