Neonatal infection is a major cause of morbidity and mortality worldwide. Increased susceptibility to infection in the neonate is attributed in part to defects in T cell-mediated immunity. A peptide:MHC class II tetramer-based cell enrichment method was used to test this hypothesis at the level of a single epitope. We found that naive T cells with TCRs specific for the 2W:I-Ab epitope were present in the thymuses of 1-d-old CD57BL/6 mice but were barely detectable in the spleen, likely because each mouse contained very few total splenic CD4+ T cells. By day 7 of life, however, the total number of splenic CD4+ T cells increased dramatically and the frequency of 2W:I-Ab-specific naive T cells reached that of adult mice. Injection of 2W peptide in CFA into 1-d-old mice generated a 2W:I-Ab-specific effector cell population that peaked later than in adult mice and showed more animalto- animal variation. Similarly, 2W:I-Ab-specific naive T cells in different neonatal mice varied significantly in generation of Th1, Th2, and follicular Th cells compared with adult mice. These results suggest that delayed effector cell expansion and stochastic variability in effector cell generation due to an initially small naive repertoire contribute to defective peptide:MHC class II- specific immunity in neonates.