We previously reported that the nude mouse-derived splenic T cell clone N-9F exhibits a proliferative response when cultured on thymic stromal cells. This N-9F proliferation is mediated by direct cell-to-cell interactions between T and thymic stromal cells. A thymic epithelial cell clone, SL10.3, also supports N-9F growth. To identify the molecule involved in T cell development in the thymus, we established mAb specific to the N-9F clone. One of these mAb, QR6.6, was found to inhibit the N-9F proliferative response on SL10.3. QR6.6-positive cells were detected in thymus but not in other lymphoid organs such as bone marrow, lymph nodes, or spleen. QR6.6-positive cells accounted for 3 to 5% of the cells in adult thymuses whereas higher percentages were found in neonatal (10-20%) and fetal thymuses (70% at E17 and 10-20% at E15). The positive cells were primarily CD4-8- thymocytes in fetuses and CD4-8- to CD4+8+ thymocytes in adults. The QR6.6 mAb precipitates a 100 kDa molecule from the N-9F clone. The addition of the mAb to fetal thymus organ culture reduces the recovery of cells at culture day 4. It was also found that the mAb inhibits fetal thymocyte proliferation on the SL10.3 thymic epithelial cell line. These results suggest that the 100 kDa molecule detected by the QR6.6 mAb may play a crucial role in the early stage of thymocyte development.