An in vitro pH-cycling experiment was completed to determine the effect of the timing of fluoride toothpaste application on de- and remineralization. Both initially sound enamel and artificially produced enamel lesions were subjected to a pH-cycling scheme. The lesions were either formed in an undersaturated calcium-phosphate-fluoride-acetate system (CPF lesions) or in a MHDP-containing lactate buffer (MHDP lesions). The two systems produced subsurface demineralization which differed in porosity, depth and surface layer characteristics. Changes in the enamel mineral content were assessed either by microhardness evaluation of the specimens at the end of the cycling period or by calcium uptake and loss during the pH-cycling period. The results show that a fluoride dentifrice treatment is very effective in inhibiting lesion formation in initially sound enamel as well as in inhibiting lesion progression. The latter was found to be primarily due to a significant decrease in mineral loss during the demineralization cycles. This observation was made irrespective of the timing of the fluoride dentifrice treatment, which was either given before or after the demineralization challenge. For the CPF lesions this inhibition was between 40 and 50%, while the corresponding value for the MDHP lesions was 15–30%. Differences between the two lesion types were also found in the dentifrice effect on lesion remineralization. In the case of the CPF lesions a small inhibition of mineral deposition was observed, while in MHDP lesions the fluoride treatment enhanced the remineralization. These observed differences are explained in terms of the differences in characteristics between these two types of lesion