Increasing expression by T-cell precursors of the T-cell antigen receptor and of the CD4 and CD8 glycoproteins during thymus development enables specific interactions between primitive T cells and thymic epithelial cells, which initiate the development of the T-cell repertoire. Given the importance of GAGs, such as HS, in cell-cell interactions in other organs, we asked whether such molecules might participate in the cellular interactions essential for the development of the thymus. Using an organ culture model in which fetal murine thymuses explanted on gestational day 14 undergo phenotypic maturation from CD3−CD4−CD8− to CD3+CD4+/CD8+, the consequences of inhibiting GAG synthesis with 6-diazo-5-oxo-norleucine (DON) were explored. Inhibition of GAG synthesis prevented elaboration of cortical thymocyte-associated HS,IL-1 and de novo expression of the TCR, CD4, and CD8. DON did not alter the expression of TCR or CD4/CD8 by mature thymocytes. Synthesis of IL-1α, TCR, CD4, and CD8 was restored by addition of HS to the cultured organs. These findings support the concept that the ontogenic (but not the constitutive) expression of the TCR and of CD4 and CD8 depends on the synthesis in the thymus of IL-1, and that IL-1 synthesis is in turn regulated by the metabolism of GAGs. Our findings suggest that components of the thymic microenvironment, particularly HS, play a critical role in the maturation of T-cell precursors.