The ability of group A streptococci to bind human plasminogen and plasmin has attracted interest, because it could provide the bacteria with a mechanism for invasion. M or M-like proteins account for the binding of several plasma proteins to group A streptococci. To investigate whether M or M-like proteins were responsible for the binding of plasminogen to group A streptococci, acid-extracted material from a type M53 streptococcal isolate was tested for its ability to bind plasminogen. Indeed, a 42-kDa plasminogen-binding protein was solubilized. Two oligonucleotides homologous with conserved sequences in known M protein genes were used as primers in the polymerase chain reaction, with chromosomal DNA from the M53 isolate. When cloned and expressed in Escherichia coli, a resulting fragment encoded a 43-kDa plasminogen-binding protein. Nucleotide sequence determination of the gene fragment revealed an open reading frame encoding a polypeptide of 43,580 Da, which matched the amino-terminal amino acid sequence of the plasminogen-binding protein extracted from M53 streptococci. The DNA sequence data also proved the relationship of the encoded protein, named PAM, to the M proteins. The plasminogen-binding domain was mapped to the amino-terminal third of PAM. Plasminogen absorbed by M53 streptococci or by immobilized PAM could be activated by streptokinase. The results provide further evidence of the diversity of the M protein family and suggest a new mechanism whereby these proteins contribute to the virulence of group A streptococci.