Caries incidence during a 2-year period was studied in 4- to 5-year-old children from three areas with contrasting levels of natural fluoride (F) in the drinking water and different regimens of topical fluoride varnish applications; group A (n = 448) was from an area with a low level of F (0.1 ppm) and semi-annual applications of fluoride varnish; group B (n = 374) was from a low F area (0.1 ppm) and no fluoride varnish treatments; group C (n = 206) was from an area with optimal F (1.2 ppm) and fluoride varnish treatments. All children were clinically assessed at baseline and after 2 years according to WHO criteria. The number of salivary mutans streptococci was estimated and scored at baseline and after 2 years with the Strip mutans method. The varnish containing 0.1% F was applied every 6 months on all accessible tooth surfaces after cleaning with a pumice paste. Basic preventive care was given to all children and restorative treatment on individual indications. Higher levels (p < 0.05) of salivary mutans streptococci were found in the low-fluoride areas compared to the optimal fluoride area at baseline and after 2 years. The caries incidence (mean dft +/- SD) in the different groups was A: 0.65 +/- 1.40; B: 1.09 +/- 1.85; C: 0.53 +/- 1.09. The difference between group B and groups A and C was statistically significant (p < 0.05). A positive relationship (p < 0.05-0.001) between salivary mutans streptococci scores at baseline and caries incidence was found in all three groups. This study confirms the close association between salivary mutans streptococci and caries incidence in preschool children and suggests a caries-reducing effect of topical applications of the fluoride silane varnish.