Cleaved, cation-derivatized Muscovite mica is utilized extensively in atomic force microscopy (AFM) imaging because of its flatness over large areas (millimeter cleavage planes with local root-mean-square roughness < 0.3 nm), ease of preparation, and ability to adsorb charged biomolecules such as DNA (work by Hansma and Laney, Guthold et al., and McMaster et al.). In particular, NiCl(2) treatment has become a common method for controlling DNA adsorption on mica substrates while retaining the mica's ultraflat surface (work by Pietrement et al.). While several studies have modeled the mica/metal ion/DNA system using macroscopic colloidal theory (DLVO, etc.; Pietrement et al., Sushko et al., Pastre et al., and Cheng et al.), nickel/mica's physicochemical properties have not been well characterized on the nanoscale. Efforts to manipulate and engineer DNA nanostructures would benefit greatly from a better understanding of the surface chemistry of nickel/mica. Here we present in situ nanometer- and attogram-scale measurements and thermodynamic simulation results that show that the surface chemistry of nickel-treated mica is more complex than generally appreciated by AFM practitioners because of metal-ion speciation effects present at neutral pH. We also show that, under certain preparations, nickel/mica allows in situ nanoscopic nucleotide sequence mapping within individual surface-adsorbed DNA molecules by permitting localized, controlled desorption of the double helix by soluble DNA binding enzymes. These results should aid efforts to precisely control the DNA/mica binding affinity, particularly at the physiological pH ranges required by enzymatic biochemistry (pH 7.0-8.5), and facilitate the development of more complex and useful biochemical manipulations of adsorbed DNA, such as single-molecule sequencing.