The Y-Ae mAb and the 1H3.1 alphabeta T cell antigen receptor (TCR) are both specific for the I-Ealpha52-68 peptide bound to the I-A(b) major histocompatibility complex (MHC) class II molecule. Antigen-presenting cells (APCs) from I-A(b+) mice with a natural or transgenic (Tg) I-Ealpha chain activate mature 1H3.1 T cells and cause the deletion of 1H3.1 TCR Tg thymocytes. However, 1H3.1 T cells were neither activated nor inactivated by confrontation with APCs from I-Ab-Ep mice in which I-A(b) molecules are occupied only by the covalently associated Ealpha52-68 peptide. Instead, immature 1H3.1 TCR Tg thymocytes were efficiently positively selected into the CD4 lineage in the I-Ab-Ep thymus. This selection relied on specific recognition of the Ealpha52-68/I-A(b) complex because it was blocked by Y-Ae. 1H3.1 TCR Tg T cells maturing in the I-Ab-Ep thymus efficiently populated the periphery, displayed a naive phenotype, and were specifically reactive to the Ealpha52-68 peptide or to I-A(b+)I-Ealpha(+) APCs, indicating that 1H3.1 T cells were not antagonized in I-Ab-Ep mice. The data identify major histocompatibility complex class II molecules with only a covalently attached self-peptide as a ligand for in vivo positive selection of T cells specific for the same peptide.