Priming a population with a pre-pandemic vaccine is being considered to maximize the response upon subsequent vaccination with a true pandemic vaccine more closely matched to the causative pandemic strain. The present study explored this prime-boost concept by evaluating different primary schedules with the pre-pandemic A/Vietnam/1194/2004(NIBRG-14) vaccine, containing 3.75 microg of HA, followed by a 6-month booster with a vaccine formulated with 3.75 microg HA of either the same strain or with A/Indonesia/05/2005(IBCDC-RG2), a heterologous strain from a different clade. In this multicentre, open, randomized study (NCT00430521) we measured immune responses in four groups (N = 48-60) of adults aged 18-60 years who received a single booster administration of either A/Indonesia/05/2005 or A/Vietnam/1194/2004 vaccine 6 months after a 1- or 2-dose (given 21 days apart) primary vaccination with A/Vietnam/1194/2004. All prime-boost schedules assessed induced early (7 days post-booster) humoral responses that met regulatory acceptance criteria. Two doses of A/Vietnam/1194/2004 given 6 months apart achieved equivalent homologous seroprotection after the second vaccination (89.6%), when compared to two doses given 21 days apart (92.7-93.2%). Remarkably, two doses of A/Vietnam/1194/2004 given 6 months apart induced a higher cross-reactive seroprotection against A/Indonesia/05/2005 (83.3%) when compared to two doses given 21 days apart (41.5-54.5%). A 6-month A/Indonesia/05/2005 booster dose after one primary dose of A/Vietnam/1194/2004 vaccine induced 92.5% seroprotection against A/Indonesia/05/2005 and 98.1% against A/Vietnam/1194/2004. Since a single booster 6 months after one primary dose of AS03-adjuvanted vaccine induces strong and rapid seroprotective immune response against both homologous and heterologous H5N1 strains, these results might have important implications for public health strategy aiming to organize vaccination campaigns with pre-pandemic vaccines.