We report a detailed analysis of TCR V alpha and V beta chain expression on immature versus mature thymocytes of normal, TCR beta-transgenic, and TCR alpha-hemizygous mice. Chain pairing between TCR V alpha and V beta chains is random on immature thymocytes, but individual chain pairs are selected in mature thymocytes. This indicates that V alpha-V beta chain pairing preferences are determined during thymic selection, and not as a result of structural constraints. Dual V alpha chain expression is found frequently on immature, but not mature thymocytes. It is not found in TCR alpha-hemizygous mice, showing that cell surface expression of dual alpha chains is caused by lack of allelic exclusion in immature thymocytes. Down-regulation of one of the alpha chains occurs concurrently with differentiation from TCRlo, CD69- to TCRhi, CD69+ phenotype, suggesting that it is associated with positive selection of the functional TCR.