The observation that nontypable (NT) Haemophilus influenzae causes serious infection in adults has stimulated interest in mechanisms that may protect the human host against NT H. influenzae infection. Incubating NT H. influenzae with normal human serum (NHS) caused dose- and time-dependent killing that varied with the individual NHS and NT H. influenzae. Adsorption of NHS with NT H. influenzae removed bactericidal activity against the adsorbing isolate but not necessarily that against others, suggesting antigenic diversity and supporting recent studies that show different outer membrane protein profiles among NT H. influenzae. Heating NHS to 56 degrees C for 30 min abolished bactericidal activity; this activity was not restored by complement-rich guinea pig serum or NT H. influenzae-adsorbed NHS. This is analogous to the "third factor" needed for intraleukocytic killing of pneumococci. Optimal opsonization of NT H. influenzae for phagocytosis by human polymorphonuclear leukocytes required antibody and complement, but other serum factors also played a role. Bactericidal activity generally, but not uniformly, correlated with opsonizing activity of individual NHS. Humoral factors may be important in host defenses against NT H. influenzae infection; their emergence during convalescence warrants further study.