Background: Invasive Haemophilus influenzae type b (Hib) infection has a high morbidity among young children, but the burden of disease and rate of Hib are different in different regions. The aim of the present study was to investigate the levels of Hib antibodies and the oropharyngeal Hib prevalence in young children. Methods: One hundred-fifty nine healthy children aged 19-36 months of age were included in this cross-sectional study. Anti- polyribosylribitol phosphate (anti-PRP) antibody concentrations were measured using commercially available enzyme-linked immunosorbent assay (ELISA), and serotyping of isolated Hib strains was conducted by slide agglutination with specific antisera. Results: Of the study participants, 57 (35.8%) were fully vaccinated (group 1A); 17 (10.7%) were incompletely vaccinated (group 1B), and 85 (53.5%) were non-vaccinated (group 2). Geometric mean titer (GMT) of anti-PRP antibody was 3.8 μg/mL, 2.2 μg/mL and 0.49 μg/mL in group 1A, group 1B and group 2, respectively. While all children in group 1 (n = 74) had seroprotective antibody concentrations (≥0.15 μg /mL), of the children in group 2 (n = 85) 31.8% did not have seroprotective anti-PRP levels (P < 0.0001). A total of 68.2% in group 2 had natural immunity. Nineteen children (33.3%) in group 1, and 46 (54.1%) in group 2 had oropharyngeal Hib colonization (P = 0.0004). Conclusions: Hib conjugate vaccine is immunogenic and reduces Hib colonization. Each country should investigate the burden of Hib disease and the natural immunity in young children, and should determine antigenic dose, number of doses administered and dose intervals before deciding whether to introduce Hib conjugate vaccine in routine immunization programs. © 2008 Japan Pediatric Society.