Distinction between lymphocyte-rich thymoma and T-lymphoblastic lymphoma/leukemia (T-LBL) can be problematic because of a predominance of precursor T cells in both, particularly if the epithelial component in a thymoma is undersampled. Because of very different clinical implications, accurate diagnosis is critical. The NOTCH1 signaling pathway is frequently activated in T-LBL and plays a central role in the pathogenesis of this disease. Antibodies to NOTCH1 intracellular domain (N1ICD), recognizing the active form of NOTCH1, have been developed. We hypothesized that detection of N1ICD would be useful in distinguishing T-LBL from thymoma and investigated a series of formalin-fixed, paraffin-embedded tissues for immunoreactivity with an N1ICD antibody using automated immunohistochemistry. Slides were scored using a 25% nuclear reactivity threshold for positivity. Hyperplastic tonsil showed positivity in few scattered interfollicular lymphoid cells, suprabasilar epithelial cells, and endothelial cells. Thymocytes from non-neoplastic thymus were largely negative for N1ICD. All thymomas tested (n=23) were negative for N1ICD, although epithelial cells and a small minority of thymocytes may be positive, requiring careful interpretation. All T-LBL cases (n=16) were scored positive for N1ICD: 8 (50%) of these showed diffuse and mostly strong immunoreactivity, whereas the remaining 8 (50%) had less extensive positivity, but with consistently >25% nuclear staining. In conclusion, normal thymocytes do not express significant levels of N1ICD. In keeping with this pattern, thymomas are negative for N1ICD, whereas a high percentage of T-LBL expresses N1ICD. Thus, N1ICD immunohistochemistry appears to be a useful method in distinguishing T-LBL from thymoma.