In situ atomic force microscopy (AFM) was used to investigate the kinetics of demineralization of human dentin collagen fibrils. Individual dentin collagen fibrils containing intrafibrillar mineral were isolated, transferred onto a glass slide and exposed to water for a day prior to demineralization studies. The fibrils then were exposed to trypsin for removal of non-collagenous proteins and subsequently demineralized in 10 vol % citric acid. Topographic images showed a gradual increase in gap-overlap depth of the fibril. The gap-overlap depth varied linearly with the square root of time before saturation at 7 nm in approximately sixty minutes, suggesting a diffusion process for demineralization of intrafibrillar mineral. Micro Raman studies of partially demineralized dentin revealed the existence of a phosphate peak at wave number close to 960 cm-1. The peak gradually disappeared in 60 minutes as the samples were exposed to 10% citric acid, supporting the notion that AFM topography may be correlated to the degree of intrafibrillar mineralization.