Previous studies have mapped amino acid contact residues of neutralizing monoclonal antibodies by sequencing in vitro neutralization escape variants, through site-specific substitution of specific charged or polar residues, and by performing binding assays with overlapping peptide libraries. We used error-prone PCR mutagenesis and yeast surface expression to identify contact residues in a high-throughput manner. Although this technique has been used previously 29 , this is the first highthroughput epitope-mapping application. By having a large panel of DIII monoclonal antibodies and selecting only variants that abolished or markedly reduced binding of a few monoclonal antibodies, we minimized the possibility that mutations altered protein folding. We have recently confirmed the recognition sites on DIII for E16 and the validity of the yeast display strategy by solving the crystal structure of the E16 Fab-DIII complex (G.N., T.O., M.D, & D.F., unpublished data).