During thymocyte development, progression from T cell receptor (TCR)beta to TCRalpha rearrangement is mediated by a CD3-associated pre-TCR composed of the TCRbeta chain paired with pre-TCRalpha (pTalpha). A major issue is how surface expression of the pre-TCR is regulated during normal thymocyte development to control transition through this checkpoint. Here, we show that developmental expression of pTalpha is time- and stage-specific, and is confined in vivo to a limited subset of large cycling human pre-T cells that coexpress low density CD3. This restricted expression pattern allowed the identification of a novel subset of small CD3(-) thymocytes lacking surface pTalpha, but expressing cytoplasmic TCRbeta, that represent late noncycling pre-T cells in which recombination activating gene reexpression and downregulation of T early alpha transcription are coincident events associated with cell cycle arrest, and immediately preceding TCRalpha gene expression. Importantly, thymocytes at this late pre-T cell stage are shown to be functional intermediates between large pTalpha+ pre-T cells and TCRalpha/beta+ thymocytes. The results support a developmental model in which pre-TCR-expressing pre-T cells are brought into cycle, rapidly downregulate surface pre-TCR, and finally become small resting pre-T cells, before the onset of TCRalpha gene expression.