The early stages of enamel surface dissolution (erosion) are of high scientific relevance; however, little is known about if and to what extent these early stages, which result in the softening of the enamel surface, are reversible. The present study investigated the possible rehardening of surface softened enamel in two separate experiments in vitro, employing physiologically relevant demineralization and remineralization times and remineralizing solutions possessing chemical properties close to those of human saliva. Atomic force microscopy (AFM) based nanoindentation has been used to study the nanomechanical properties and the ultrastructure of polished enamel samples as affected by demineralization/remineralization cycles. In contradiction to previous studies employing microhardness techniques, no enamel rehardening has been observed after remineralization; however, the demineralization schemes used in the present study (short treatments with a citric acid buffer) are not comparable to the ones used in previous studies, making a comparison difficult. A mineral deposition after remineralization on softened enamel samples has been observed by AFM; however, a protective effect of this deposited layer could not be revealed. The exposure of softened enamel samples to either a remineralizing solution or a mineral water led to an improved acid resistance of these samples as shown in a demineralization/remineralization cycle. The present study has demonstrated that AFM based nanoindentation is a useful tool to investigate the demineralization and remineralization of surface softened enamel with high accuracy.