The phagocytosis of Haemophilus influenzae type b (Hib) by rat macrophages and the intracellular fate of ingested organisms was investigated using an acridine orange-crystal violet assay. There was a correlation between the ability of organisms to survive in macrophages in vitro and their ability to cause invasive disease. Encapsulated Hib survived and replicated within macrophages, whereas capsule-deficient mutants, although more susceptible to phagocytosis, were killed after ingestion. Differences in lipopolysaccharide also affected the ability of encapsulated Hib to survive in macrophages. The presence of viable intracellular organisms in macrophages in vivo may enhance the persistence of bacteremia and may also be important in mediating the entry of Hib into the central nervous system.