The humoral immune system in higher vertebrates is unique in its ability to generate highly diverse antibody responses against most pathogens as well as against certain malignancies. Several technologies have been developed to exploit this vast source of potentially therapeutic antibodies, including hybridoma technology, phage display and yeast display. Here, we present a novel, high-throughput technology (the Symplex Technology) for rapid direct cloning and identification of human antigen-specific high-affinity antibodies from single antibody-producing cells of immune individuals. The utility of the technology was demonstrated by isolation of diverse sets of unique high-affinity antibodies against tetanus toxoid and influenza virus from immunized volunteers. Hence, the Symplex Technology is a new method for the rapid isolation of high-affinity antibodies directly from humans.