CD97 is an activation-induced antigen on leukocytes which belongs to a new group of seven-span transmembrane (7-TM) molecules, designated EGF-TM7 family. Family members, including EMR1 and F4/80, are characterized by an extended extracellular region with several N-terminal epidermal growth factor-like (EGF) domains. Alternative splicing of CD97 results in isoforms possessing either three (EGF1, 2, 5), four (EGF1, 2, 3, 5) or five EGF domains (EGF1, 2, 3, 4, 5). We recently identified decay accelerating factor (DAF, CD55), a regulatory protein of the complement cascade, as a cellular ligand of the smallest isoform. Employing mutants of CD97(EGF1, 2, 5) in which the EGF domains have been systematically deleted, we here demonstrate the necessity of at least three tandemly linked EGF domains for the interaction with CD55. Consistent with the involvement of different EGF domains, monoclonal antibodies directed against the first EGF domain as well as the removal of Ca2+, for which binding sites exist in the second and fifth EGF domain, blocked binding to CD55. Compared to CD97(EGF1, 2 ,5) the larger isoforms CD97(EGF1, 2, 3, 5) and CD97(EGF1, 2, 3, 4, 5) have a significantly lower affinity for CD55. Thus, alternative splicing may regulate the ligand specificity of CD97 and probably other members of the EGF-TM7 family.