The fluoride concentration in cows' milk has been reported to vary with the fluoride levels in drinking water but it seldom exceeds 0.5 microg/ml. This raised a question as to whether any caries-protective effect could be attributed to the intrinsic fluoride of milk. Two samples of cows' milk with intrinsic fluoride concentrations of 0.03 and 0.3 microg/ml, respectively, were assessed for their protective effect on enamel in an in vitro demineralization model at relatively severe and mild acidic challenges (pH 4.6 and 5.0, respectively). Polished enamel discs were incubated individually in 5.0 ml of demineralization solution for 20 h per day alternated with 1-hour incubations in 1.0 ml of milk or control buffers: group 1, demineralization solution only (negative control); group 2, milk with 0.03 microg/ml fluoride; group 3, milk with 0.03 microg/ml fluoride; supplemented with NaF to 0.3 microg/ml fluoride; group 4, milk with 0.3 microg/ml fluoride; group 5, 0.3 microg/ml fluoride in 20 mM HEPES, pH 6.7; group 6, milk with 0.03 microg/ml fluoride supplemented with NaF to 5.0 microg/ml fluoride (positive control). The solutions were renewed each day and the calcium concentration in the demineralization solutions was followed during 4 days. The results showed that the protective effect of intrinsic milk fluoride on enamel is limited by the severity of the acidic challenge: There was a significant inhibition of the demineralization in groups 3-6 compared to groups 1 and 2, but only at pH 5.0 (p<0.0001) and not at pH 4.6 (p = 0.2). The organic components of milk had limited protection against demineralization because milk and HEPES with the same fluoride concentration gave similar results. The 36% reduction in calcium loss at pH 5.0 by treatment with milk with only 0.3 microg/ml fluoride is an indication that intrinsic milk fluoride has some caries-protective properties.