We examined the virulence role of group B Streptococcus (GBS) beta-hemolysin/cytolysin (beta h/c) in a neonatal-rabbit model of GBS pulmonary infection. Rabbits infected intratracheally with wild-type (wt) GBS developed focal pneumonia and, by 18 h after infection, had 100-fold more bacteria in lung tissue than did rabbits infected with a delta beta h/c mutant. Mortality (40% vs. 0%), development of bacteremia, and mean bacterial blood counts were all significantly higher in the rabbits challenged with wt GBS than in those challenged with the delta beta h/c mutant. Lung compliance during mechanical ventilation was impaired after injection of wt GBS but not after injection of the Delta beta h/c mutant strain. This work, to our knowledge, provides the first in vivo evidence for a critical role of the beta h/c toxin in GBS neonatal pneumonia and in the breakdown of the pulmonary barrier to systemic infection.