NK1.1+ T cells represent a specialized T cell subset specific for CD1d, a nonclassical MHC class I-restricting element. They are believed to function as regulatory T cells. NK1.1+ T cell development depends on interactions with CD1d molecules presented by hematopoietic cells rather than thymic epithelial cells. NK1.1+ T cells are found in the thymus as well as in peripheral organs such as the liver, spleen, and bone marrow. The site of development of peripheral NK1.1+ T cells is controversial, as is the nature of the CD1d-expressing cell that selects them. With the use of nude mice, thymectomized mice reconstituted with fetal liver cells, and thymus-grafted mice, we provide direct evidence that NK1.1+ T cells in the liver are thymus dependent and can arise in the thymus from fetal liver precursor cells. We show that the class I+ (CD1d+) cell type necessary to select NK1.1+ T cells can originate from TCRalpha-/- precursors but not from TCRbeta-/- precursors, indicating that the selecting cell is a CD4+CD8+ thymocyte. 5-Bromo-2'-deoxyuridine-labeling experiments suggest that the thymic NK1.1+ T cell population arises from proliferating precursor cells, but is a mostly sessile population that turns over very slowly. Since liver NK1.1+ T cells incorporate 5-bromo-2'-deoxyuridine more rapidly than thymic NK1.1+ T cells, it appears that liver NK1.1+ T cells either represent a subset of thymic NK1.1+ T cells or are induced to proliferate after having left the thymus. The results indicate that NK1.1+ T cells, like conventional T cells, arise in the thymus where they are selected by interactions with restricting molecules.