Children previously unvaccinated against influenza are recommended to receive two doses of trivalent inactivated influenza vaccine (TIV). This study assessed the age limit for a second dose of TIV in previously unvaccinated children. Two hundred and twenty-four children were immunized with TIV (Influsplit SSW/Fluarix; GlaxoSmithKline Biologicals). Subjects aged 6-9 years (n=110) received two doses of TIV 4 weeks apart; those aged 10-13 years (n=114) received a single dose. Serum haemagglutination inhibiting (HI) antibody titers were evaluated before and after each vaccination. CHMP criteria for HI antibody response were met for all three vaccine strains after one vaccine dose in children 10-13 years and after two doses in children 6-9 years. A marked benefit was observed in 8-year-old children after a second dose even though one dose would be sufficient against all strains. Our results provide additional evidence that Influsplit SSW/Fluarix is well tolerated and highly immunogenic in children and support recommendations that previously unvaccinated children aged <9 years should receive two doses of influenza vaccine.