Negative selection during T cell ontogeny involves selective induction of apoptosis in thymocytes. In peripheral lymphoid cells, apoptosis may be mediated via the APO-1 pathway. Here we report that APO-1 is constitutively expressed on the vast majority of human thymocytes but down-regulated at a mature stage of thymocyte development (TCR(hi)). This stage of development is characterized by CD28hi, CD44hi, CD69hi and up-regulation of Bcl-2 protein. We define a new thymocyte subpopulation that expresses high levels of APO-1 and intermediate levels of T cell receptor alpha/beta (TCR(im)/APO-1hi). The TCR(im)/APO-1hi population contains a large fraction of dead cells, suggesting that the APO-1 pathway may be involved in negative selection of at least a fraction of thymocytes after intrathymic activation.