Mouse NK1+ T cells constitute a subset of alpha/beta TCR+ T cells that specialize in the rapid production of cytokines, in particular IL-4, and may promote the differentiation of Th2-type CD4 T cells. Their TCRs, like those of a homologous subset of human T cells, use an invariant TCR alpha chain and were recently shown to be specific for the beta 2-microglobulin-associated, MHC class I-like CD1 molecules, which are encoded outside the MHC. In contrast to mainstream thymocytes, which recognize their positively selecting MHC ligand on thymic epithelial cells, positive selection of NK1+ T cells requires their CD1 ligand to be expressed on bone marrow-derived cells. To investigate the nature of the bone marrow-derived cell involved, chimeric mice were constructed with tissues from normal, SCID, and MHC-deficient mice, so that CD1 could be selectively expressed by different subsets of bone marrow-derived cells in the thymus. CD1 expression was also directly assessed using an anti-CD1 mAb, and a CD1-specific T cell hybridoma. The results suggest that immature (CD4+8+ double-positive) cortical thymocytes are the source of CD1 presentation for positive selection of NK1+ T cells.