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The aim of this study was to analyze the potential influence of long-term exposure in subinhibitory concentrations of chlorhexidine on the emergence of tolerant and/or persistent cells in oral streptococci. The two oral streptococcal isolates <i>S. mutans</i> ATCC25175 and <i>S. sobrinus</i> ATCC33402 were incubated, after long-term subinhibitory exposure to chlorhexidine, in liquid growth media containing high concentrations of chlorhexidine. A distinct subpopulation of more chlorhexidine-tolerant cells could be detected in streptococci that had been previously exposed to subinhibitory concentrations of chlorhexidine but not in the control strains. These more biocide-tolerant and persisting microbial subpopulations might also arise in vivo. Therefore, the rational and proper use of antimicrobials in dentistry, especially when used over a long period of time, is crucial.