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