W e investigated the role of serum bactericidal activity in Hemophiplus influenzae type b infections in infants with meningitis and in a rat model. In infected infants, 13/22 admission sera had bactericidal activity against the infecting strain, and bacteremia was as frequent in those with bactericidal activity (54%) as those without (56%). The coexistence of bactericidal activity and bacteremia was reproduced and studied in experimentally infected weanling rats. Serum from such rats kills in vitro 95% of conventionally broth-grown bacteria within 10 min, but does not kill organisms obtained from the infected animals. Thus bactericidal activity as conventionally determined for H. influenzae b may have no relevance in vivo, Incubation of broth-grown bacteria in normal rat serum for 30 min at 37 degrees C produces a resistance like that of in vivo organisms. This phenotypic conversion depends on factors that are of molecular weight less than 1,000, stable to 100 degrees C, but destroyed by ashing. When injected intravenously into nonimmune animals, broth-grown bacteria are quickly cleared, while serum-preincubated bacteria are not. The latter, however, are cleared when injected into bacteremic rats (half-life 30 min). Bacteremia in the rats may persist despite this capacity for clearance because bacteria are entering the blood from extravascular fluids, which contain greater than 90% of the total bacterial burden.