Streptococcus mutans is the main pathogenic agent of dental caries. Glucosyltransferases (Gtfs) produced by these bacteria are important virulence factors because they catalyze the extracellular synthesis of glucans that are necessary for bacterial accumulation in the dental biofilm. The diversity of GtfB and GtfC isozymes was analyzed in 44 genotypes of S. mutans that showed a range of abilities to form biofilms in vitro. Several approaches were used to characterize these isozymes, including restriction fragment length polymorphism analysis of the gtfB and gtfC genes, zymographic analysis of the identified GtfB and GtfC genotypes, and quantitation of isozyme production in immunoblot experiments with specific monoclonal antibodies. A high diversity of gtf genes, patterns of enzymatic activity, and isozyme production was identified among the isolates tested. GtfC and, to a lesser extent, GtfB were produced in significantly higher amounts by strains that had high biofilm-forming ability than by strains with low biofilm-forming ability. Biofilm formation was independent of the GtfB and GtfC genotype. Atypical strains that showed an apparent single Gtf isozyme of intermediate size between GtfB and GtfC were also identified. The results indicate that various expression levels of GtfB and GtfC isozymes are associated with the ability of distinct S. mutans genotypes to grow as biofilms, strengthening the results of previous genetic and biochemical studies performed with laboratory strains. These studies also emphasize the need to identify factors that control gtf gene expression.