This nationwide study assessed the antimicrobial susceptibility and seroprevalence of Streptococcus pneumoniae in paediatric carriage isolates and in clinical isolates from adult pneumococcal disease in Greece during the years 2004-2006. Among 780 isolates recovered from the nasopharynx of children <6 years old attending day-care centres, non-susceptibility rates to penicillin, cefuroxime, ceftriaxone, erythromycin, tetracycline and trimethoprim/sulfamethoxazole were 34.7%, 25.1%, 1.0%, 33.5%, 26.4% and 44.2%, respectively. Among 89 adult clinical isolates, the respective rates were 48.3%, 46.1%, 5.6%, 48.3%, 32.6% and 40.4%. High-level resistance to penicillin, cefuroxime and ceftriaxone was recorded for 14.4%, 23.3% and 0.1% of paediatric carriage isolates, whereas for clinical adult isolates the respective rates were 25.8%, 38.2% and 2.2%. No resistance to levofloxacin and moxifloxacin was recorded, although 3.5% of paediatric carriage isolates and 23.2% of adult clinical isolates had minimum inhibitory concentrations of ciprofloxacin >2mg/L. Serotypes 19F, 14, 23F and 6B were the most prevalent among carriage and clinical isolates. The 7-valent pneumococcal conjugate vaccine was estimated to provide coverage against 71.7% of paediatric carriage isolates and 51.3% of adult clinical isolates. Resistance rates among clinical isolates from adult sources were higher than those recorded among paediatric carriage S. pneumoniae isolates and displayed an increasingly resistant profile compared with previous reports from our country, warranting continuous vigilance.