By screening a genomic lambda library of Streptococcus equi subsp. zooepidemicus, we have cloned and sequenced a gene, termed fnz, encoding a fibronectin (Fn)-binding protein called FNZ. On the basis of the deduced amino acid sequence of FNZ, the mature protein has a molecular mass of approximately 61 kDa. Analysis of FNZ reveals a structural organization similar to that of other cell surface proteins from streptococci and staphylococci. The Fn-binding activity is localized to two domains in the C-terminal part of FNZ. One domain is composed of five repeats, which contain a motif similar to what has earlier been found in other Fn-binding proteins in streptococci and staphylococci. The first and second repeats are separated by a short stretch of amino acids, including the motif LAGESGET, which is an important part of the second Fn-binding domain. This motif is also present in an Fn-binding domain (UR) in protein F of Streptococcus pyogenes. A fusion protein covering the Fn-binding domain of FNZ inhibits the binding of the 29-kDa N-terminal fragment of Fn to cells of various streptococcal species as well as to Staphylococcus aureus.