The site of negative selection in the thymus has been inferred from a range of different experiments. Analysis of thymic deletion of V beta 5+, V beta 11+ or V beta 17a+ cells in H-2E transgenic mice led to the theory that negative selection occurs predominantly in the medulla (specifically, through presentation by medullary dendritic cells). Other experiments investigated whether transgenic TCR are deleted at the double-positive (DP) or single-positive stage following encounter with peptide ligand: by flow cytometric analysis deletion is generally found to occur at the DP thymocyte stage and as these cells are found predominantly in the cortex, it has been inferred that this is the key site of negative selection. The visualization of apoptotic thymocytes in situ has recently been reported for specific examples of negative selection. Using a panel of TCR transgenic lines in which negative selection occurs at different stages of thymocyte development, we have used TUNEL staining to analyse the anatomical sites of thymocyte apoptosis. For the first time we have been able to compare directly the sites of deletion induced by the endogenous cognate peptides or by endogenous superantigen. We show that generalization from the medullary deletion of V beta 5+, V beta 11+ or V beta 17a+ cells by the endogenous superantigens Mtv 8 and 9 from limited examples of cortical deletion by exogenous peptide administered to TCR transgenic mice is over-simplified. Apoptotic thymocytes in mice lacking Mtv superantigens are indeed localized in the cortex. However, when deletion is induced by cognate self peptide, apoptosis can occur in the cortex, the medulla or at the junction between the two.