Having previously demonstrated the feasibility of administering A/H5N1 and seasonal influenza vaccine antigens in an MF59-adjuvanted tetravalent formulation, we now report on long-term antibody persistence and responses to a booster dose of a combined seasonal-pandemic, tetravalent influenza vaccine in adults. The primary objective was the evaluation of responses to a booster dose of tetravalent influenza vaccine containing seasonal (A/H1N1, A/H3N2, and B) and avian (A/H5N1, clade 2) influenza virus strains administered to 265 healthy 18- to 40-year-old volunteers 1 year after priming with one or two clade 1 A/H5N1 doses. Secondary objectives were assessment of reactogenicity, safety, and antibody persistence 1 year after priming with a combined seasonal-pandemic, tetravalent vaccine. Responses to seasonal strains met all European licensure criteria; seroprotection rates were 94 to 100%, 100%, and 61 to 90% for A/H1N1, A/H3N2, and B strains, respectively. Anamnestic responses were observed against homologous and heterologous A/H5N1 strains whether priming with one or two A/H5N1 doses, with a monovalent A/H5N1 vaccine, or with a tetravalent vaccine. A single dose of MF59-adjuvanted A/H5N1 vaccine given alone or as part of a fixed combination with a seasonal influenza vaccine was sufficient to prime adult subjects, resulting in robust antigen-specific and cross-reactive antibody responses to heterologous booster immunization 1 year later. These data support the feasibility of incorporating prepandemic priming into seasonal influenza vaccination programs. (This study has been registered at clinicaltrials.gov under registration no. NCT00481065.).