The development of antibodies is a major defense mechanism against viruses. Understanding the repertoire of antiviral antibodies induced during infection is a necessary prerequisite to defining the protective activities of an antiviral antibody response. The isolation of antigen specific memory B cells and subsequent stimulation to antibody producing cells provides a powerful tool to study the antibody repertoire in infected individuals. We have used this approach to analyze the antibody repertoire against glycoprotein B (gB) of human cytomegalovirus (HCMV), a major antigen for the induction of antiviral antibodies during infection and a constituent of experimental vaccines in humans. We find in different infected individuals that the vast majority of gB-specific B cells produce antibodies that cannot neutralize free virus. Antibodies with antiviral capacity target two domains of gB that have not been previously identified. The identification of these new antigenic domains was possible with the aid of a 3D molecular model of HCMV gB. Our results will be useful for vaccine development since comparison of the immune response after natural infection with that induced by vaccination can be readily accomplished. Moreover, neutralizing human monoclonal antibodies could constitute powerful therapeutics to combat the infection in populations at risk for HCMV disease.