Group B Streptococcus (GBS) is a Gram-positive bacterium that causes sepsis and meningitis in neonates and infants. Although several GBS-associated virulence factors have been described, the mechanisms of GBS invasive disease are not well understood. To characterize additional virulence factors, a novel in vitro infection assay was developed using rat fetal lung explants. However, application of GBS to the system induced rapid lung tissue destruction associated with increased media acidity. Since lactic acid produced by other streptococci is an important virulence factor, we hypothesized that lactic acid contributed to the virulence of GBS. Spent growth media and neutralized-spent media were applied to explants and results indicated that neutralization of the media completely protected the tissue from degradation. These results were verified using multiple viability assays and with transformed cell lines. Furthermore, comparable spent media from Escherichia coli did not induce tissue cytotoxicity, suggesting that GBS produces organic acids in excess of other potential bacterial pathogens. Analysis of the spent media indicated that l-lactate levels reached approximately 70 mM, indicating that lactic acid is a major constituent of the metabolic acid produced by GBS. Treatment of explants with lactic acid alone produced dose-dependent tissue degradation, indicating that lactic acid is independently sufficient to induce target-tissue cytotoxicity. Finally, both spent media and 23.6 mM lactic acid produced dramatic tissue autofluorescence; the basis for this is currently unknown. These studies demonstrate that GBS-produced lactic acid is a potential virulence factor and may contribute to GBS invasive disease.