Peripheral T lymphocytes each express surface T cell receptor (TCR) alpha and beta chains of a single specificity. These are produced after random somatic rearrangements in TCR alpha and beta germline genes. Published model systems using mice expressing TCR alpha and/or beta chain transgenes have shown that allelic exclusion occurs conventionally for TCR-beta. TCR alpha chain expression, however, appears to be less strictly regulated, as endogenous TCR alpha chains are often found in association with transgenic TCR beta chains in TCR alpha/beta transgenic mice. This finding, coupled with the unique structure of the TCR alpha locus, has led to the suggestion that unlike TCR beta and immunoglobulin heavy chain genes, TCR alpha genes may make multiple rearrangements on each chromosome. In the current study, we demonstrate that the majority of TCR-, noncycling thymocytes spontaneously acquire surface expression of CD3/TCR. Further, we show that cultured immature thymocytes originally expressing specific TCR alpha and beta chains may lose surface expression of the original TCR alpha, but not beta chains. These data provide evidence that not only must multiple rearrangements occur, but that TCR alpha gene rearrangement continues even after surface expression of a TCR alpha/beta heterodimer, apparently until the recombination process is halted by positive selection, or the cell dies. Sequential rearrangement of TCR alpha chain genes facilitates enhanced production of useful thymocytes, by increasing the frequency of production of both in-frame rearrangements and positively selectable TCR alpha/beta heterodimers.