Objective: The study aimed to assess the caries-inhibiting effect of EC40 (a chlohexidine varnish, CHX) on pits and fissures of first permanent molars of 6-7-year-old children. Methods: 6-7-year-old children were selected from schools in Wuhan, PR China. To be included in the study, each child had to have two sound contra-lateral first permanent molars and at least 3 decayed primary molars. A spilt mouth design was used in this study. The test tooth received CHX application on occlusal surfaces and pits and control tooth received no CHX. These children were randomly assigned to different groups. Group A children (n=150) received EC40 application at baseline, 6, 12 and 18 months. Group B children (n=155) received EC40 application at baseline, 3, 12 and 15 months. Caries was scored at baseline and two years later using fiber-optic light. Results: The caries increment in pits and fissures of each pair (CHX molar-control molar) was compared after two years. In group A, 203 pairs both the experimental and the control molar were caries free, in 27 pairs, experimental molars exhibited caries, in 18 pairs control molars had caries and in 7 pairs both the experimental and the control molar had caries. In group B, 236 pairs were caries free, in 28 pairs, experimental molars exhibited caries, in 21 pairs control molars had caries and in 13 pairs both the experimental and the control molar had caries. The lower caries increment on CHX molars as compared to control molars appeared to be not statistically significant (Sign test for pairs). Conclusion: The present schedules of application of EC40 did not decrease caries increment in pits and fissures of first permanent molars of children aged 8-9 years who at baseline exhibited at least 3 decayed primary molars.