Human monoclonal antibodies (HuMAbs) against HR1a from Protobothrops (formerly Trimeresurus) flavoviridis venom were obtained by the fusion of SP2/0-Ag14 myeloma cells and spleen cells from KM mice immunized with purified HR1a. The ability of HuMAbs to neutralize the HR1a was determined by in vitro neutralization assay and by neutralization of the hemorrhagic activity. The initial screening of over 300 hybridoma fusion wells resulted in the establishment of 80 HR1a-reactive hybridomas. Of the reactive clones, HuMAb HR1a-7 and HR1a-18 neutralized both proteolytic and hemorrhagic activity of HR1a. Mapping of epitope recognized by the reactive clones was performed by using an ELISA that measured antibody binding to overlapping peptides (15 amino acid peptide offset frameshifted by three residues) covering the metalloproteinase domain sequence of HR1a. HuMAbs HR1a-7 and HR1a-18 neutralized HR1a by reacting with peptides of EQQRYLNNFRFIELV and IVNTLNETYRYL. The three-dimensional structure of HR1a based on a homology modeling predicted that these two epitopes are surface exposed.