Group B streptococci (GBS) are the leading cause of pneumonia and sepsis in human newborns. Exudative pulmonary edema and alveolar hemorrhage seen in GBS pneumonia indicate vascular damage, and we reported that GBS injure lung microvascular endothelial cells (LMvEC) both in vivo and in vitro. The specific GBS factors causing LMvEC injury are uncertain, but GBS beta-hemolysin activity is associated with lung epithelial cell injury. We hypothesized that GBS beta-hemolysin contributes to LMvEC injury and exudative pulmonary edema. To test this hypothesis we used isogenic nonhemolytic and hyperhemolytic GBS mutants derived by transposon insertional mutagenesis from three different wild-type strains. Hemolytic titers for each strain were calculated using live GBS and Tween 80/starch-stabilized extracts of log-phase GBS. All nonhemolytic mutants lacked detectable hemolytic activity, whereas hyperhemolytic mutants produced 4-16 times the hemolytic activity of their parent strains. LMvEC injury was assayed by light microscopy, the release of lactate dehydrogenase, trypan blue nuclear staining and Evans blue-albumin flux. Compared with the parent strains, all nonhemolytic mutants caused significantly reduced, and all hyperhemolytic mutants caused significantly greater lactate dehydrogenase release from and trypan blue nuclear staining of LMvEC. Moreover, a nonhemolytic mutant caused reduced and a hyperhemolytic mutant caused increased Evans-blue albumin flux across polar LMvEC monolayers. These findings were corroborated by light microscopic evidence of hemolysin-associated damage to the LMvEC monolayers. We conclude that GBS beta-hemolysin promotes LMvEC injury and increases permeability in vitro, and speculate that GBS beta-hemolysin contributes to the pathogenesis of alveolar edema and hemorrhage in early onset GBS pneumonia.