The interaction between the anaphylatoxin C5a and its receptor involves two distinct sites. One site is formed by acidic residues at the receptor N-terminus and contributes to only ligand binding. The second site, responsible for activation, is less well defined. In this study, we demonstrate that the receptor residue D282, near the extracellular face of transmembrane domain VII, is a component of the second ligand-binding site. Mutation of D282 to A decreases the sensitivity of the receptor to activation by intact C5a but not by its less potent metabolite, C5adR74, which lacks the C-terminal arginine74. The mutation of the R74 residue of C5a to A causes a 60-fold decrease in wild-type receptor sensitivity, but only a 2-fold decrease for the receptor mutated at D282. In contrast, the mutation of R74 to D makes C5a completely inactive on both wild-type and A282 C5a receptors. The mutation of D282 to R partly restores the response to C5a[D74], which is a more effective ligand than C5a at the mutant receptor. A peptide mimic of the C5a activation domain with a C-terminal R potently activates the wild type but is only a weak agonist at the mutant D282R-C5a receptor. Conversely, a peptide with D at the C-terminus is a more effective activator of D282R than wild-type C5a receptors. These data indicate that the R74 side chain of C5a makes an interaction with receptor D282 that is responsible for the higher potency of intact C5a versus that of C5adR74.