A 16,600-D outer membrane protein is present in all strains of Haemophilus influenzae and antibodies to this protein are present in human serum. This study was designed to assess the role of this outer membrane protein (P6) in nontypeable H. influenzae as a target for human serum bactericidal antibody. P6 was isolated and coupled to an affinity column. Depleting normal human serum of antibodies to P6 by affinity chromatography resulted in reduced bactericidal activity of that serum for nontypeable H. influenzae. Immunopurified antibodies to P6 from human serum were bactericidal. Finally, preincubation of bacteria with a monoclonal antibody that recognizes a surface epitope on P6, inhibited human serum bactericidal killing. Taken together, these experiments establish that P6 is a target for human bactericidal antibodies. This observation provides evidence that P6 plays a potentially important role in human immunity to infection by nontypeable H. influenzae.