This study describes the effects of varnishes containing 0%, 10%, 20%, or 40% chlorhexidine diacetate on the microflora of human fissure dental plaque. Sandarac, a natural resin, was used as the varnish base. Ten subjects, each with at least four sound fissures harboring high levels of Streptococcus mutans, participated in the study. The fissures in each of the individuals were randomly assigned to four experimental groups, in each of which one of the varnishes was tested. The varnish treatment consisted of a single application of a small amount of varnish onto the fissures. Apart from the selected fissures, the rest of the dentition was left untreated. All chlorhexidine-containing varnishes selectively suppressed S. mutans in fissure plaque, and had no effect on total viable counts or on the numbers of Actinomyces viscosus/naeslundii and Streptococcus sanguis beyond one week. The extent of the suppression depended upon the concentration of chlorhexidine in the varnish, 40% chlorhexidine varnish giving the greatest suppression of S. mutans. At 22 weeks, after a single treatment with varnish containing 40% chlorhexidine, mean S. mutans counts were more than ten times lower than in the control or 10%-chlorhexidine varnish group. At that time, S. mutans was still undetectable in five out of ten experimental fissures in this group. The results suggested that sandarac varnishes containing high concentrations of chlorhexidine can be used successfully for long-term suppression of S. mutans in dental fissures.