Purpose: To evaluate the in vitro effect of different application frequencies of dentifrices containing CPP-ACP and fluoride on enamel demineralisation inhibition using a pH cycling model. Materials and Methods: One hundred twenty blocks of human enamel were divided into 8 groups according to the treatment and number of times that the dentifrice slurry was applied (3 or 5 times). Control: dentifrice without fluoride (CO3 and CO5); fluoride dentifrice (FD3 and FD5, commercial dentifrice 1100 ppm as NaF); MI Paste (MP3 and MP5, Recaldent) and MI Paste Plus (MPP3 and MPP5, Recaldent 900 ppm as NaF). After pH cycling, cross-sectional microhardness (CSH) measurements were taken. The demineralised enamel changes were analyzed on three blocks per group by MicroCT. Data were analyzed by ANOVA and Tukey's HSD post-hoc test (p = 0.05). Results: Statistically significantly higher CSH values (p 0.05) were obtained for the surface layers (25 and 50 µm) for FD3, FD5, MP3 and MP5. The MP and FD groups showed similar results and had the least mineral loss. The MP increased the mineral density in enamel and decreased the depth of the lesion. Conclusion: Inhibition of subsurface enamel demineralisation is possible with MP and FD, independent of the application frequencies. However, the MP and MPP groups had higher mineral density when five applications were performed. Keywords: casein, dentifrice, fluoride, prevention