We are investigating a nontypeable Haemophilus influenzae (NTHI) strain, R2866, isolated from a child with meningitis. R2866 is unusually resistant to killing by normal human serum. The serum 50% inhibitory concentration (IC50) for this strain is 18%, approaching that of encapsulated H. influenzae. R3392 is a derivative of R2866 that was found to have increased sensitivity to human serum (IC50, 1.5%). Analysis of tetrameric repeat regions within lipooligosaccharide (LOS) biosynthetic genes in both strains indicated that the glycosyltransferase gene lgtC was out of frame ("off") in most colonies of R3392 but in frame with its start codon ("on") in most colonies of the parent. We sought antigenic and biochemical evidence for modification of the LOS structure. In a whole-cell enzyme-linked immunosorbent assay, strain R3392 displayed reduced binding of the Galalpha1,4Gal-specific monoclonal antibody 4C4. Mass spectrometry analysis of LOS from strain R2866 indicated that the primary oligosaccharide glycoform contained four heptose and four hexose residues, while that of R3392 contained four heptose and three hexose residues. We conclude that the R2866 lgtC gene encodes a galactosyltransferase involved in synthesis of the 4C4 epitope, as in other strains, and that expression of lgtC is associated with the high-level serum resistance that has been observed for this strain. This is the first description of the genetic basis of high-level serum resistance in NTHI, as well as the first description of LOS composition in an NTHI strain for which the complete genome sequence has been determined.