The mouse monoclonal antibody MR6 recognizes a 200 000 MW protein (gp200-MR6), which is expressed highly on human thymic cortical epithelial cells. The antigen is also expressed on some epithelial tumours and we have previously shown that MR6 inhibits the proliferation of the colon carcinoma cell lines HT29. However, the role of this molecule in the thymus is not known. In order to generate reagents that could be used in murine thymic functional studies we isolated antibodies specific to human gp200-MR6, using a phage display library expressing single-chain (sFv) antibodies. Three independent clones were isolated by panning with purified protein and their specificity was confirmed by immunohistochemistry, Western blotting and flow cytometry. In addition to human thymus, these phage antibodies also recognized the homologous antigen in mouse, pig and other species. Expressed as soluble sFv one of these clones inhibited the proliferation of HT29 cells and a mouse thymic epithelial cell line, suggesting that this antibody exhibits similar functional activity to MR6. In fetal thymic organ culture, thymocytes recovered from thymic lobes cultured in the presence of this sFv, were reduced in number fivefold compared with the control and the majority remained at the double-negative stage of development. These data indicate that gp200-MR6 plays an important role in thymocyte development. In addition, this is the first report to demonstrate that specific sFv can be used to study, and alter, thymic development. This work also highlights the advantage of phage antibody technology in selecting such reagents for functional assays.