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SUMMARY: Studies on the pathogenesis of Haemophilus influenzae b infection have used bacteria grown in vitro which are relatively serum-sensitive (using serum devoid of anticapsular antibody) compared to organisms taken from infected hosts. We compared the virulence of relatively serum-sensitive and serum-factor induced serum-resistant H. influenzae b by inoculating rats with organisms having one or the other phenotype. The serum-resistant phenotype was more virulent following intraperitoneal or intravenous inoculation; however, there was no difference in the incidence of colonization or bacteraemia following intranasal inoculation. Furthermore, organisms colonizing the pharynx of rats had the serum-resistant phenotype. Thus, different phenotypes of the same strain of H. influenzae b differed in virulence following parenteral, but not intranasal, inoculation of bacteria. This could be explained by a change from serum-sensitive to serum-resistant phenotype shortly after entering the nasopharynx. The phenotype of micro-organisms grown in vitro may differ from organisms in infected individuals and these differences may be of critical importance in studies of immunity to infection and the pathogenesis of infection.