We have previously shown that phenotypic allelic exclusion of TCR alpha-chain is functional only in mature thymocytes. A significant proportion of immature thymocytes (TCRlow) express more than one cell surface alpha-chain, but mature thymocytes (TCRhigh) show phenotypic allelic exclusion and express only a single alpha-chain. We have analyzed thymocytes for both surface and intracellular alpha-chain expression and find that the majority of mature thymocytes express a second alpha-chain intracellularly. This result is predicted by a model in which the developmentally regulated allelic exclusion of the TCR alpha-chain is caused by competition between alpha-chains for the beta-chain rather than by models in which one alpha-chain is down-regulated or in which selection favors cells with only a single alpha-chain species. Changes in the relative amounts of alpha- and beta-chains available for pairing may therefore allow competition between the two alpha-chains for the beta-chain. Peripheral T cells also frequently express second alpha-chains in the cytoplasm (18-27%), despite a rather low frequency of dual alpha-chain expression on the cell surface (2-4%). The frequency of nonsurface expressed alpha-chains is reduced somewhat compared with thymocytes, indicating that an additional level of control of allelic exclusion operates during the maturation of peripheral T cells.