Streptococcus agalactiae or group B Streptococcus (GBS) is a gram-positive bacterium that causes significant infant morbidity and mortality (1). Vaccine conjugates comprising capsular polysaccharides and protein antigens are currently under development (4). Analysis and characterization of streptococcal antigens for vaccine development have suffered from a lack of standardized techniques to determine subcellular localization. We describe a method to fractionate GBS into its subcellular components to determine the localization of protein and polysaccharide antigens. This information can be used to study mechanisms of protein and polysaccharide expression in these organisms and ultimately to help identify useful targets for vaccine development. The subcellular fractionation of GBS used methods originally developed for the gram-positive coccus