Apolipoprotein E (apoE), a 299 amino acid protein, is a ligand for the low density lipoprotein receptor (LDLr). It has been established that basic amino acids situated between apoE residues 136 and 150 participate in the interaction of apoE with the LDLr. Evidence suggests that apoE is heterogeneous on lipoproteins in its conformation and in its ability to react with cell surface receptors. Our goal was to produce mAbs that could serve as conformational probes of the LDLr binding site of apoE. We used a series of apoE variants that have amino acid substitutions at residues 136, 140, 143, 144, 145, 150, 152, and 158 to identify the epitopes of two anti-human apoE monoclonal antibodies (mAbs), 1D7 and 2E8, that inhibit apoE-mediated binding to the LDLr. We show that most of the variants that have reduced reactivity with the LDL receptor also have reduced reactivity with the mAbs. The epitopes for both mAbs appear to include residues 143 through 150 and thus coincide with the LDLr-binding site of apoE. It is notable that mAb 2E8, but not 1D7, resembles the LDLr in showing a reduced reactivity with apoE (Arg158 --> Cys). While most of the receptor-defective variants involve replacement of apoE residues directly implicated in binding, substitution of Arg158 by Cys is thought to indirectly affect binding of apoE to the LDLr by altering the conformation of the receptor-binding site. To determine whether the similarity in specificities of the mAbs and the LDLr reflect structural similarities, we cloned and characterized the cDNAs encoding the light and heavy chains of both mAbs. Primary sequence analysis revealed that, although these two antibodies react with overlapping epitopes, their respective complementarity determining regions (CDRs) share little homology, especially those of their heavy chains. The two mAbs, therefore, likely recognize different epitopes or topologies within a limited surface of the apoE molecule. Four negatively charged amino acids were present in the second CDR of the 2E8 heavy chain that could be approximately aligned with acidic amino acids within the consensus sequence of the LDLr ligand-binding domain. This could indicate that mAb 2E8 and the LDLr use a common mode of interaction with apoE.