Objectives: To determine the level of mutans streptococci in children after partaking in two-time toothbrushing program at school. Methods: 70 kindergarten children aged 3-5 yr. in the remote area of Thailand had participated in this study. They were assigned to brush their teeth under the supervision of their class teachers; 1 time brushing after lunch for the control group, and 2 times brushing at 8.00 am and after lunch for the experimental group. Mutans streptococci levels in saliva were determined at 4 occasions as followings: at baseline, after 1 month, after 6 months and after 8 months. Paraffin-stimulated whole saliva was collected individually using spatula method as described by Kohler and Bratthall, and then cultivated on Mitis-Salivarius Bacitracin agar in 5% CO2 at 37C for 48 h. The level of mutans streptococci grown was scored into 3 categories: 1 = 0-20 CFU, 2 = 21-100 CFU and 3 = >100 CFU. Results: At baseline, the levels of mutans streptococci of both control and experimental groups were not different (Mann-Whitney U test; p-value > 0.05). Compared to the baseline, no significant difference in mutans streptococci levels was observed in both groups even though following up to 6 months. Interestingly, after 8 months, the level of mutans streptococci in the experimental group decreased significantly compared to that at the baseline (Wilcoxon Sign Rank test; p-value < 0.01). In the control group, however the level of the organisms remained at the same level as the baseline. Conclusion: The study clearly shows that two-time toothbrushing program can be introduced as a simple method to reduce such cariogenic agent as mutans streptococci so as to promote good oral hygiene in children. (Supported by the grant from Mahidol University 2001)