Thymocyte maturation consists of a number of stages, the goal of which is the production of functioning T cells that respond to foreign antigenic peptides using their clonotypic receptors. Selection of a productively rearranged TCR beta-chain is the first stage in the process and occurs at the double-negative to double-positive (DP) transition. Later maturation stages are based on changes in markers such as CD5, CD69, or IL-7R. A stage in which a-chains are selected has also been identified using beta-chain transgenic mice. Here we identify two additional selection stages in human thymocytes based on characteristics of the TCR. alpha selection is measured directly by identification of in-frame rearrangements and is associated with the appearance of CD3 on the DP thymocyte surface. The next stage has not yet been described and involves selection of thymocytes that express shorter TCR beta-chain complementarity-determining region 3 (CDR3). This stage is associated with the acquisition of high levels of CDR3 by DP cells and the transition to SP thymocytes. The extent of CDR3 length selection observed is a function of the TCR V and J genes. We propose that CDR3 length selection is based on recognition of the MHC. Thus, there exist limitations on the allowable length of that portion of the TCR most intimately in contact with MHC and peptide. This may be a physical representation of positive selection.