The application of non-contact optical profilometry for non-destructive study of changes in the surface roughness of natural enamel surfaces during in vitro demineralization was investigated. Repeated measurement of profiles of the same enamel surfaces after successive demineralization episodes was achieved by a kinematic specimen mount that could be removed, then accurately repositioned. Surface roughness parameters and reflectivity of natural enamel surfaces exposed to either a demineralizing solution (test) or deionized water (control) for up to 6 days were measured at 6- to 24-hour intervals. The results prior to demineralization showed that surface roughness varies with position on the enamel surface. During demineralization there was an approximately linear increase in surface roughness for the first 70 h followed by somewhat erratic behaviour, and a saturating exponential with time increase in reflectivity for the first 70 h which subsequently remained constant. Changes in enamel surface during in vitro demineralization were also observed using scanning electron microscopy.