The fear of malevolent use of variola virus by terrorists has led to the implementation of a health care worker vaccination program and to the consideration of vaccination for the general public. However, due to concerns about side effects of the classical smallpox vaccine, especially for immunocompromised individuals, a safer vaccine is urgently needed. We characterized the immunogenicity of modified vaccinia virus Ankara (MVA), one of the more promising alternative smallpox vaccines, in a cohort of 10 chronically HIV-1-infected individuals undergoing highly active antiretroviral therapy (HAART). Nine subjects received smallpox vaccination as children while one subject was never vaccinated against smallpox. All the subjects had CD4 counts >400 cells/mm(3) and 8 out of 10 had undetectable viral loads. MVA was able to elicit humoral and cellular immune responses in the majority of individuals. Vaccinia-specific antibodies were mainly of the IgG class while T cells specific to vaccinia were predominantly CD8(+). The immune responses were maintained over 1 year. Similar vaccinia specific humoral immune responses were observed when our cohort of HIV-1-infected individuals was compared to smallpox-vaccinated healthy subjects. The observed immune responses suggest that the highly attenuated MVA could be used as a substitute vaccine against smallpox in chronically HIV-1-infected individuals undergoing HAART.