A DNA vaccine encoding Ag85A from Mycobacterium tuberculosis was administered to guinea pigs by epidermal gene gun bombardment and its protective efficacy was determined. Vaccination with Ag85A DNA twice significantly reduced the severity of pulmonary pathology and number of pulmonary colony-forming units (CFU) (p<0.01). When immunogenic synthetic Ag85A peptide was used as a booster, lung pathology was improved significantly and pulmonary CFU were reduced dramatically. Neither Ag85A DNA nor BCG Tokyo protected the guinea pigs from hematogenous spread of tubercle bacilli to the spleen because splenic granulomas without central necrosis were recognized. When the vaccinated guinea pigs were followed up for 7 months, the pulmonary lesions became fibrotic in guinea pigs vaccinated with Ag85A DNA plus Ag85A peptide, or BCG Tokyo, and no tubercle bacilli were detected. The protective efficacy of the tuberculosis Ag85A DNA vaccine was improved significantly by peptide boosting. It is concluded that dosage and peptide boosting are important in the induction of higher protective efficacy by a tuberculosis DNA vaccine.