Inactivation of fibronectin (Fn) binding by insertional mutagenesis of Streptococcus sanguis with Tn916 reduces virulence of this bacterium in the rat model of infective endocarditis (IE). Transconjugants were screened for Fn adherence using an ELISA adherence test. One transconjugant had a decreased adherence to immobilized Fn. Southern hybridization demonstrated that the insertion occurred only once in this mutant. The parent strain and mutant strain JL113 were used as challenge strains in a rat endocarditis model. These experiments demonstrated that the mutant had a reduced ability (P less than 0.05) to produce IE. Spontaneous excision of Tn916 from JL113 produced strains identical to both the parental and mutant phenotypes. One strain (JLR-19) that retained the mutant phenotype and one (JLR-15) that regained the parental phenotype for Fn binding were tested for their ability to produce IE. These strains demonstrated that the ability to bind Fn and to produce IE were correlated after Tn916 excision. The reduced virulence of the mutant suggested that adherence of S. sanguis to immobilized Fn plays an important role in the production of IE.