During characterization of the surface antigens of serotype III group B streptococci (GBS), a protein with an apparent M(r) of approximately 173,500 migrating on a SDS--polyacrylamide gel was found to have an N-terminal amino acid sequence identical to that of the plasmin receptor (Plr) of group A streptococci, a surface-localized glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This work begins to characterize GBS GAPDH and to assess its functional activity on the cell surface. The 1.0-kb gapC gene of GBS was amplified by PCR. plr and gapC demonstrated 87% homology. An anti-Plr monoclonal antibody reacted with GBS whole cells, suggesting GBS GAPDH is surface localized. Multiple serotypes of GBS demonstrated functional GAPDH on their surfaces. The anti-Plr monoclonal antibody recognized GBS protein bands of approximately 41 and 173.5 kDa, by Western blot. Presumably, these represent monomeric and tetrameric forms of the GAPDH molecule. GBS GAPDH was demonstrated by Western blot analysis to interact with lys- and glu-plasminogens. Fluid-phase GBS GAPDH interacted, by means of ELISA, with immobilized lys-plasminogen, glu-plasminogen, actin, and fibrinogen. Enzymatically active GAPDH, capable of binding cytoskeletal and extracellular matrix proteins, is expressed on the surface of GBS.