CONTEXT: There is renewed interest in use of smallpox vaccine due to the potential for a bioterrorist attack. This would involve vaccinating health care workers who were previously vaccinated. OBJECTIVE: To evaluate the use of diluted vaccinia virus in vaccination of previously vaccinated (non-naive) participants. DESIGN, SETTING, AND PARTICIPANTS: Eighty non-naive participants, aged 32 to 60 years, were randomized in a single-blinded study to receive either undiluted or diluted (1:3.2, 1:10, or 1:32) doses of smallpox vaccine. A comparison group, aged 18 to 31 years, of 10 vaccinia-naive participants received undiluted vaccine. Participants were enrolled between April 1 and May 15, 2002, at the National Institute of Allergy and Infectious Diseases Vaccine and Treatment Evaluation Unit at Saint Louis University, St Louis, Mo. INTERVENTION: Smallpox vaccine was administered by scarification using 15 skin punctures in the deltoid region of the arm. MAIN OUTCOME MEASURES: Presence of a major reaction, defined as a vesicular or pustular lesion or area of palpable induration surrounding a central lesion following vaccination, and measures of viral shedding and antibody titers. RESULTS: Initial vaccination resulted in a major reaction in 64 of 80 non-naive participants. Ninety-five percent of non-naive participants had major reactions in the undiluted group, 90% in the 1:3.2 dilution group, 81% in the 1:10 dilution group, and 52.6% in the 1:32 dilution group. All (n = 10) of the vaccinia-naive participants had major reactions. Compared with vaccinia-naive participants, non-naive participants had significantly smaller skin lesions (P =.04) and significantly less incidence of fever (P =.02). Preexisting antibody was present in 76 of 80 non-naive participants. Antibody responses were significantly higher and occurred more rapidly in the non-naive participants compared with the vaccinia-naive participants (P =.002 for day 28 and P =.003 for 6 months). Vaccinia-naive participants shed virus from the vaccination site 2 to 6 days longer and had significantly higher peak mean viral titers when compared with the non-naive participants (P =.002). CONCLUSIONS: Previously vaccinated persons can be successfully revaccinated with diluted (<or=1:10) smallpox vaccine. Fewer adverse reactions were observed in this study of non-naive participants when compared with events in vaccinia-naive participants, which may be due to immunologic memory.