Streptococcus gordonii genes involved in beta-glucoside metabolism are induced in vivo on infected heart valves during experimental endocarditis and in vitro during biofilm formation on saliva-coated hydroxyapatite (sHA). To determine the roles of beta-glucoside metabolism systems in biofilm formation, the loci of these induced genes were analyzed. To confirm the function of genes in each locus, strains were constructed with gene inactivation, deletion, and/or reporter gene fusions. Four novel systems responsible for beta-glucoside metabolism were identified, including three phosphoenolpyruvate-dependent phosphotransferase systems (PTS) and a binding protein-dependent sugar uptake system for metabolizing multiple sugars, including beta-glucosides. Utilization of arbutin and esculin, aryl-beta-glucosides, was defective in some mutants. Esculin and oligochitosaccharides induced genes in one of the three beta-glucoside metabolism PTS and in four other genetic loci. Mutation of genes in any of the four systems affected in vitro adhesion to sHA, biofilm formation on plastic surfaces, and/or growth rate in liquid medium. Therefore, genes associated with beta-glucoside metabolism may regulate S. gordonii in vitro adhesion, biofilm formation, growth, and in vivo colonization.