T lymphocytes eradicate and provide long-term immunity to infections caused by intracellular pathogens. The mechanisms that determine in vivo T cell response sizes are poorly understood. Although it is speculated that the relative processing efficiency of different epitopes determines the hierarchy of T cell responses following immunization, this hypothesis has not been rigorously tested. We therefore mutagenized the secreted p60 Ag of Listeria monocytogenes to alter the efficiency of T cell epitope generation. Ag-processing efficiencies in cells infected with the different L. monocytogenes mutants ranged from one H2-K d-associated p60 217–225 epitope generated per 15 intracellularly degraded p60 molecules (1/15) to one epitope per 350 degraded p60 molecules (1/350), ie, a spectrum encompassing a 20-fold range of efficiencies. Mice infected with L. monocytogenes secreting inefficiently processed p60 (1/350) did not mount p60 217–225-specific T cell responses. However, increasing the efficiency of Ag processing by a factor of 5 to 1/70 restored the T cell response size to normal, while further increases in the efficiency of p60 217–225 generation to 1/50, 1/35, and 1/17 did not further augment specific T cell responses. Our studies demonstrate an Ag-processing threshold for in vivo T cell activation. Surprisingly, once this threshold is achieved, further enhancement of Ag-processing efficiency does not enhance the size of T cell responses.