Sialyl Tn (sTn) is a mucin-associated carbohydrate antigen expressed in most types of human adenocarcinoma. Defining the configuration of tumor cell surface sTn recognized by antibodies is important for understanding the basis for the cancer cell specificity of sTn-reactive mAbs, for the development of more effective mAbs, and for designing cancer vaccines against sTn. In this study, we compared the immunogenicity of synthetic single sTn disaccharide epitopes and clusters [sTn(C)] of 3 sTn epitopes covalently linked via serine to keyhole limpet hemocyanin [KLH; sTn-KLH and sTn(C)-KLH, respectively]. The cell surface sTn configurations were analyzed with the use of sera from mice immunized with these neoglycoproteins and a panel of sTn-reactive mAb. Sera from mice immunized with sTn-KLH reacted in direct and inhibition assays with sTn-human serum albumin (HSA) but only weakly with sTn(C)-HSA, whereas sera from mice immunized with sTn(C)-KLH reacted with sTn(C)-HSA but not with sTn-HSA. Both anti-sTn and anti-sTn(C) sera reacted with ovine submaxillary mucin (a natural source of sTn) and with sTn-positive human tumor cell line LS-C but not with sTn-negative LS-B cells. With regard to the sTn-reactive mAbs, B72.3 reacted exclusively with clustered sTn, whereas mAb B195.3R11 reacted preferentially with unclustered sTn. Results with mAbs TKH2, B239.1, and CC49 were less clear, although all reacted more strongly with clustered sTn than with unclustered sTn. These results suggest that sTn is recognized at the tumor cell surface in at least two quite distinct configurations, as clustered and nonclustered arrays.