To initiate an investigation into the biochemistry and mechanism of group B β-hemolytic Streptococcus virulence, bacterial cultures grown in suspension were centrifuged, and the bacteria and media were subjected to extensive fractionation. Each fraction was assayed for physiologic activity by repeated intravenous infusion into adult unanesthetized sheep. Pulmonary artery pressure, arterial PO2, and rectal temperature were monitored. The media fraction, but not the bacteria, contained a component (molecular weight, 2 X 106) composed of 84% carbohydrate and 16% protein with physiological activity. Two mg quantities, when infused, caused the pulmonary artery pressure to increase 100%, PO2 to decrease by 20% and chills and fever. The active component could be degraded by hot phenol-water extraction into a pure polysaccharide (molecular weight, 200,000). This lower molecular weight polysaccharide retained the identical physiologic properties when infused in the sheep. The degraded component precipitated with group B-specific antiserum. This study demonstrates that, in the sheep, a pure polysaccharide is the physiologically active part of a high-molecular-weight toxin synthesized by group B β-hemolytic Streptococcus type III and that this component has a different carbohydrate composition from the group B capsular antigen. Speculation: The clinical syndrome associated with group B β-hemolytic Streptococci in early onset disease is caused by the interactions of an extracellular bacterial component and a specific target tissue in the infected infant.