Human CR2 (CD21, EBV receptor) is an approximately 145-kDa receptor and a member of the regulators of complement activation gene family. Regulators of complement activation proteins are characterized by the presence of repeating motifs of 60 to 70 amino acids that are designated short consensus repeats (SCR). CR2 serves as a receptor for four distinct ligands. Three of these ligands (complement C3, gp350/220 of EBV, and CD23) interact with the amino terminal 2 of 16 SCR (SCR 1 and 2). Previous studies have determined that at least four sites are important in allowing CR2 to efficiently bind EBV. Two of these sites are also important for binding mAb OKB7, a reagent that blocks both EBV and iC3b/C3dg binding to CR2. We have identified and characterized important sites of iC3b ligand binding by utilizing human-mouse CR2 chimeras, a rat anti-mouse CR2 mAb designated 4E3 that blocks receptor binding to C3, and human CR2-derived peptides. In addition to demonstrating an important role for the same sequence in SCR 1 that is part of the mAb OKB7 and EBV binding site, we have identified a new region within SCR 2 that interacts with C3. These results, when compared with a model of a dual SCR solution structure derived from human factor H SCR, predict that two distinct largely surface-exposed sites on CR2 interact with iC3b. A relative twist of 130° about the long axis of the second SCR in this model would be necessary for these sites to form a single patch for iC3b binding on CR2.