Knock-out mutants of Streptococcus gordonii Challis were constructed and assayed for binding to extracellular matrix proteins (EMPs) by enzyme-linked immunosorbent assay (ELISA). It was shown that (i) the mutant lacking the cell wall polysaccharide receptor could no longer bind type I and type II collagen, (ii) the mutant lacking the fibronectin-binding proteins CshA and FbpA was also strongly impaired in collagen binding and (iii) the mutant lacking the methionine sulfoxide reductase MsrA was significantly impaired in fibronectin binding. Our results indicate that binding to EMPs by S. gordonii is a multifactorial process controlled by genes located at three different chromosomal sites.