The potential to increase the supply of vaccine by diluting the vaccinia virus of Lister strain to face possible bioterrorism with smallpox was evaluated. Vaccinia-naïve subjects (n=97) were randomized to receive either undiluted or diluted (1:5, 1:10) vaccine, and previously vaccinated subjects (n=122) were randomized to receive either undiluted or diluted (1:10, 1:30) vaccine. Except two subjects who received 1:30 diluted vaccine, the vaccination of all subjects was successful clinically. All subjects had significant vaccinia-specific T cell and antibody responses. The diluted vaccine was not associated with decreased local or systemic reactions, lower T cell responses, or higher antibody titers when compared with undiluted vaccine. Here we show the diluted vaccine of Lister strain can be used in vaccinia-naïve subjects and previously vaccinated subjects if viral titer ≥10⁸ and 10^7.5pfu/mL after dilution, respectively. The reactogenicity of vaccinia virus may not be a dose-dependent response.