The process of thymocyte differentiation occurs within the context of the thymic microenvironment, in which T cell precursors interact with thymic microenvironmental cells and extracellular matrix. Here we studied the expression of galectin-3, a g -galactoside binding lectin, in the thymus of young adult mice. Galectin-3 was found mainly in the medulla and to a lesser extent in the cortex. We further showed that distinct microenvironmental elements, such as thymic epithelial cells, the epithelial component of thymic nurse complexes and phagocytic cells of the thymic reticulum produce, secrete and accumulate galectin-3 on the cell surface. Functionally, galectin-3-enriched medium inhibited in vitro thymocyte interactions with thymic microenvironmental cells, accelerated the release of thymocytes from thymic nurse cells and inhibited the reconstitution of these lymphoepithelial complexes. These effects were blocked by exogenous lactose (Gal g 1–4Glc), but not melibiose (Gal § 1–6Glc), and by a monospecific anti-galectin-3 antibody. Recombinant galectin-3 also inhibited thymocyte/thymic epithelial cell interactions. Our data indicate that intrathymically produced galectin-3 disrupts thymocyte/microenvironmental cell interactions, thus acting as a deadhesion molecule.