We questioned whether strains of ampicillin-resistant, non-beta-lactamase-producing (AmpR NBLP) Haemophilus influenzae with lower affinity penicillin-binding proteins (PBPs) might have altered virulence. The virulence of resistant transformant strains and the susceptible recipient was compared using infant rats. Following intraperitoneal inoculation, there was a significantly lower mortality rate and incidence and magnitude of bacteremia with two of three transformants compared to the recipient strain. Reduced virulence was not associated with greater bactericidal activity of serum or human neutrophils or faster clearance of the transformant following intravenous injection. Heated rat or human plasma supported exponential growth of the recipient, but not the transformant, suggesting deficient in vivo multiplication. We conclude that H. influenzae with altered PBPs are less virulent in an infant rat model which may be related to differences in in vivo growth.