CD97 is an EGF‐TM7 receptor found on various carcinomas where expression levels correlate with dedifferentiation and tumor stage, smooth muscle cells and leukocytes. CD97 acts as an adhesion molecule by binding to its cellular ligand, CD55. In this study, we demonstrate that 2 immunodominant CD97 epitopes are not equally present in the various cell types. Differences were apparent in gastrointestinal tumors and smooth muscle cells where monoclonal antibodies (mAbs) to the first epidermal growth factor (EGF) domain (CD97^EGF) showed a more restricted staining pattern than mAbs to the stalk region (CD97^stalk). This discrepancy was not detectable in cultured gastrointestinal tumor cell lines. In fact, the selection of the CD97 mAb influences the result of clinical studies. Thus, we clarified the reason(s) for these differences in CD97 mAb staining on various cell types. We provide evidence that epitope accessibility for CD97^EGF mAbs depends on N‐glycosylation. Immunoprecipitation of CD97 from the Colo 205 tumor cell line revealed the established 78 and 83 kDa products, while a 52 and 57 kDa band were obtained from smooth muscle cells. N‐glycosidase F reduced the size of CD97 in Colo 205 cells to 52–57 kDa. Culturing these cells with tunicamycin resulted in the same decrease in size and impaired CD97^EGF mAb binding. As shown by site‐directed mutagenesis, deletion of the N‐glycosylation sites located within the EGF domains efficiently disturbed CD97^EGF mAb immunoreactivity and, importantly, binding of CD55. In conclusion, CD97^EGF epitope accessibility for mAbs and ligand binding is influenced by cell type‐specific N‐glycosylation. © 2004 Wiley‐Liss, Inc.