Precursor T cells in the thymus are contained within a subpopulation of thymocytes that lack the markers CD4 and CD8^1–5. We have examined the heterogeneity of these cells by flow cytometric analysis, and defined four subpopulations using the cell surface markers Thy-1, J11d^6,7 and the IL-2 receptor (IL-2R). The J11d⁺ subset of CD4⁻8⁻ cells all bear the antigen Thy-1, and some express the IL-2R. Staining and RNA analysis of J11d⁺ cells suggest that some express receptors of the CD3γδ type, but none express CD3αβ receptors. In fetal thymus organ culture, the J11d⁺ cells diversify to form 'cortical type' CD4⁺8⁺ cells and 'medullary type' cells expressing either CD4 or CD8; in vivo they repopulate the thymus of an irradiated host and seed the periphery with T cells. In contrast, the J11d⁻ subset of CD4⁻8⁻ thymocytes do not all bear Thy-1 and none express the IL-2R, but some express antigen receptors of the CD3αβ type. They have more limited diversification potential in organ culture, and in vivo fail to recolonize the irradiated host in a homing-independent assay. We conclude that they are not precursor T cells, but rather a side-branch from the main line of T cell differentiation.