Objective: The aim of this in vitro preliminary study was to assess the effect of smokers’ saliva (assuming their higher calcium concentration) in combination with fluoridated toothpaste on the enamel uptake of alkali-soluble (KOH-soluble) fluoride. Materials and methods: Four enamel slabs were cut from each of 14 impacted third molars and randomly assigned into 4 groups. Unstimulated saliva samples were collected from two age and sex matched volunteers. One of the samples was taken from a heavy smoker and the other sample was taken from a non-smoker. Two groups (A and B) were shaken in saliva (A in smoker’s saliva, B in nonsmoker’s saliva) for 5 min and then shaken for 3 min in a toothpaste/deionized water slurry (1:3 w/w). One of the groups (group C) received no saliva treatment and was only shaken in toothpaste slurry for 3 min. The treatment was repeated after a 6-hour period. One of the groups (D) served as a control group with no treatment. Results: Calcium concentration in the smoker’s saliva was higher than in the nonsmoker’s saliva. The enamel uptake of KOH-soluble fluoride in group A was significantly higher than that in the other two treatment groups, B and C. The enamel uptake of KOH-soluble fluoride in all 3 groups was statistically different from that in the control group. Conclusion: These results demonstrate that saliva collected from a heavy smoker, which had higher salivary calcium concentration, enhances enamel uptake of alkali-soluble fluoride and encourages us to conduct a large-scale study.