This study describes the effects of varnishes containing 0, 25, 33 and 40% chlorhexidine diacetate on mutans streptococci in human approximal dental plaque. The chlorhexidine release from the varnishes was determined in vitro. Eleven subjects participated in the clinical experiment, each with at least five approximal areas harboring high levels of mutans streptococci. The approximal areas in each of the individuals were randomly assigned to five experimental groups, in each of which one of the varnishes was tested; 40% chlorhexidine varnish was tested in two experimental groups. The varnish treatment consisted of a single application of a small amount of varnish onto the selected approximal areas. From one of the sites receiving the 40% chlorhexidine varnish, all visible varnish was removed 15 min after application. The volunteers were asked to leave the varnish on the remaining treated sites and not to brush their teeth for 8 h. All chlorhexidine varnishes strongly suppressed mutans streptococci until 4 months after the varnish application. The extent of the suppression depended upon the concentration of chlorhexidine in the varnish, 40% chlorhexidine varnish giving the greatest suppression of mutans streptococci. No significant difference was found between the numbers of mutans streptococci from sites where the 40% varnish was removed after 15 min and sites where the 40% chlorhexidine varnish was left. The results suggested that 40% chlorhexidine varnish can be used successfully for the long-term suppression of mutans streptococci. A contact time of the varnish with the tooth surface of only 15 min is sufficient to achieve this long-term suppression.