Glass ionomer cement with addition of chlorhexidine used as a varnish on tooth surfaces has been shown to reduce the number of inter-proximal mutans streptococci (ms). The effect of a single application of such a varnish containing 2.5% chlorhexidine on occlusal caries development of the first permanent molars on 6-year-old children in a high caries incidence area was investigated. The children were examined according to WHO criteria and 262 children with 2 caries free contralateral molars were selected for treatment. Salivary ms samples were collected using the Strip-mutans (SM) method. After brushing the occlusal surfaces with a toothbrush and pumice in water slurry, rinsing and drying with a cotton roll, glass ionomer cement containing chlorhexidine (GI-CHX) and glass ionomer (GI) were applied randomly with a micro brush and the varnish was covered with occlusal wax. At baseline, the mean defs was 18.18 and DMFS was 0.25 and after one year 18.24 and 0.83 respectively. The salivary ms scores were high or very high (SM 2 and 3) in 85.6% at baseline. At the one-year follow up, the GI-CHX and GI materials could not be detected in the fissures. Also, a large number of fissure sealants had been placed in the molars outside the study protocol; thus 4% of the GI-CHX and GI, and 70% of the untreated were sealed at year one. Overall, there was no significant difference between the caries-reducing effect of GI-CHX and GI, but a trend towards a higher effect was seen for GI-CHX. Excluding the sealed molars the reduction was 74% in the GI-CHX-group, and 71% in the GI-group. Conclusion: Addition of 2.5% chlorhexidine to glass ionomer did not seem to increase the caries-reducing effect of the varnish in this high caries incidence population.