Expression of a single Ag receptor on lymphocytes is maintained via allelic exclusion that generates cells with a clonal receptor repertoire. We show in normal mice and mice expressing functionally rearranged TCR alphabeta transgenes that allelic exclusion at the TCR alpha locus is not operational in immature thymocytes, whereas most mature T cells express a single TCRV alpha-chain. TCRV alpha allelic exclusion in mature thymocytes is regulated through a CD45 tyrosine phosphatase-mediated signal during positive selection. Using functional and genetic systems for selection of immature double TCRV alpha+ thymocytes, we show that peptide-specific ligand recognition provides the signal for allelic exclusion, i.e., mature T cells maintain expression of the ligand-specific TCRV alpha-chain, but lose the nonfunctional receptor. Whereas activation of TCRV beta-chains or CD3epsilon leads to receptor internalization, TCRV alpha ligation promotes retention of the TCR on the cell surface. Although both TCRV alpha- and TCRV beta-chains trigger phosphotyrosine signaling, only the TCRV beta-chain mediates membrane recruitment of the GTPase dynamin. These data indicate that TCRV alpha-directed signals for positive selection control allelic exclusion in T cells, and that developmental signals can select for single receptor usage.