Despite the expression of only one type of receptor, there is great variation in the ability of different cell types to discriminate between C5a and its more stable metabolite, C5a des Arg74. The mechanism that underlies this phenomenon is not understood but presumably involves differences in the interaction with the C5a receptor. In this paper, we have analyzed the effects of a substitution mutation of the receptor (Glu199 → Lys199) and the corresponding reciprocal mutants (Lys68 → Glu68) of C5a, C5a des Arg74 and peptide analogues of the C-terminus of C5a on the ability of the C5a receptor to discriminate between ligands with and without Arg74. The use of these mutants indicates that the Lys68/Glu199 interaction is essential for activation of receptor by C5a des Arg74 but not for activation by intact C5a. The substitution of Asp for Arg74 of C5a [Lys68] produces a ligand with equal potency on both the wild-type and mutant receptors, suggesting that it is the C-terminal carboxyl group rather than the side chain of Arg74 that controls the responsiveness of the receptor to Lys68. In contrast, the mutation of Lys68 to Glu68 has little effect on the ability of either C5a or C5a des Arg74 to displace [125I]C5a from the receptors, indicating that binding of ligand and receptor activation are distinct but interdependent events. C5a and the truncated ligand, C5a des Arg74, appear to have different modes of interaction with the receptor and the ability of the human C5a receptor to discriminate between these ligands is at least partly dependent on an interaction with the receptor residue, Glu199.