Most invasive Haemophilus influenzae type b strains possess a duplication of the capsulation locus. Further amplification resulting in as many as 5 copies has been described. To verify whether amplification is involved in vaccine failure, the number of copies of the locus was determined by Southern blotting in 90 strains from children with true vaccine failure (TVF) between 1993 and 1999 and in 139 strains from unvaccinated children (50 collected between 1993 and 1999 and 89 collected between 1991 and 1992, before routine immunization was introduced). A significantly greater proportion of strains from TVFs contained multiple copies, compared with strains from control children (24% vs. 10%; P = .0379), which suggests that amplification of the capb locus may be a contributory factor in vaccine failure. The presence of multiple-copy strains was associated with disease other than meningitis.