Clinical studies suggest that the long-term suppression of mutans streptococci on tooth surfaces after intensive chlorhexidine therapy is mainly due to bacterial interference. Other streptococci and also Actinomyces naeslundii are proposed to inhibit regrowth of mutans streptococci after suppression by the agent. We have tested this hypothesis in gnotobiotic rats associated with Streptococcus mutans alone, or associated with S. mutans and strains of Streptococcus oralis, Streptococcus sanguis, Streptococcus gordonii, Streptococcus mitis biovar I, and A. naeslundii. Left lower jaws in these rats were treated with concentrated chlorhexidine varnish, and the return of S. mutans on the treated jaws monitored. In mono-associated rats, S. mutans regained the level of the untreated right lower jaw in approximately 1 week. In contrast, S. mutans remained suppressed for several weeks in rats multi-associated with other streptococci and actinomyces strains. The suppression was more pronounced in the rats fed on basal diet with little free sugars than in rats fed on a sucrose-containing diet. Counts of other streptococci recovered quickly from the intensive chlorhexidine treatment, but A. naeslundii remained suppressed for at least 1 week. The findings demonstrate the crucial importance of the oral microflora in controlling regrowth of mutans streptococci after chemotherapy.