Human cytomegalovirus (HCMV) is a ubiquitous herpes virus that causes fatal diseases in immunocompromised patients. There are many limitations on the treatment of HCMV disease, and the development of the preventive modality in the HCMV infection is necessary. One of preventive strategies would be obtained by the utilization of DNA-mediated immunization with plasmid carrying an antigen-encoding gene. This study was performed to evaluate the efficiency of the premedication methods that could induce neutralizing antibodies in BALB/c mice, which were injected with plasmid encoding HCMV gB. BALB/c mice were divided into 4 groups and pretreated with bupivacaine, cardiotoxin, gold and sham, respectively. Antibody titers were monitored by immunofluorescent assay, and neutralizing antibody was measured by plaque reduction assay. IgM and IgG seroconversion were found in all the tested mice. IgG antibodies appeared at 2 weeks postinoculation, reached peak levels at 6 weeks postinoculation and persisted over 6 months in all groups. The geometric mean of the peak 1gG antibody titers in each group was 1:448, 1:416, 1:208, and 1:88, respectively. Neutralizing antibody was developed, and the percent residual infectivity in 1:200 diluted sera at 8 weeks postinoculation was 24%, 34%, 43%, and 61%, respectively. These results suggested that DNA vaccine using the gene encoding HCMV gB with bupivacaine pretreatment is one of candidate methods for developing immunity to HCMV.