The relationship between the numbers of salivary mutans streptococci and the acid production in dental plaque after a single application of the 40% chlorhexidine varnish EC40 has been studied. Thirteen healthy subjects were treated with EC40 varnish. Saliva samples were taken before and up to 12 weeks after treatment to count mutans streptococci and lactobacilli. At the same time points plaque samples were taken before and after sucrose challenge and analyzed for protein and organic acid. Suppression of salivary mutans streptococci was observed together with a reduced production of lactic acid in sucrose-challenged dental plaque in 9 subjects while inhibition of acid production without significant suppression of mutans streptococci was observed in the other 4 participants. The duration of the effects differed among the individuals but never exceeded 6 weeks. We conclude that a prolonged suppression of mutans streptococci and acid production was not achieved by a single treatment with EC40 varnish in all subjects. Moreover, reduced acidogenicity of dental plaque after chlorhexidine treatment was not necessarily predicted by suppression of mutans streptococci in saliva.