We measured the force required to peel single-stranded DNA molecules from single-crystal graphite using chemical force microscopy. Force traces during retraction of a tip chemically modified with oligonucleotides displayed characteristic plateaus with abrupt force jumps, which we interpreted as a steady state peeling process punctuated by complete detachment of one or more molecules. We were able to differentiate between bases in pyrimidine homopolymers -peeling forces were 85.3 ±4.7 pN for polythymine and 60.8±5.5 pN for polycytosine, substantially independent of salt concentration and the rate of detachment. We developed a model for peeling a freely jointed chain from the graphite surface and estimated the average binding energy per monomer to be 11.5±0.6 k B T and 8.3±0.7 k B T in the cases of thymine and cytosine nucleotides. The equilibrium free-energy profile simulated using molecular dynamics had a potential well of 18.9 k B T for thymidine, showing that non-electrostatic interactions dominate the binding. The discrepancy between the experiment and theory indicates that not all bases are adsorbed on the surface or that there is a population of conformations in which they adsorb. Force spectroscopy using oligonucleotides covalently linked to AFM tips provides a flexible and unambiguous means to quantify the strength of interactions between DNA and a number of substrates, potentially including nanomaterials such as carbon nanotubes.Interactions of polyelectrolytes with solid substrates have several important applications in materials science and engineering. 1, 2 On complexation with neutral particles, polyelectrolytes convert these particles into charged species, enabling dispersions in aqueous media, and find use in detergents, cosmetics, gels, food additives, and oil recovery. In a biological context, our interest is in the interaction between single and double-stranded DNA (ssDNA and dsDNA) and substrates such as graphite and carbon-nanotubes (CNTs) that can potentially play a vital role in biomedicine, 3, 4 nanotechnology, 5,6 and relevant for the understanding of the origin of life. 7 Importantly, ssDNA has been used successfully for dispersion and solution-based manipulation of CNTs -ssDNA forms a stable hybrid with a nanotube by wrapping around the CNT in helical fashion. 8,9 The hybrid is useful for dispersion, sorting, 8,9 and patterned placement of nanotubes, 6 for transportation of DNA into cells, and for killing cancer cells by thermal ablation. 3 Strength of dispersion, ability to sort, and stability in the cellular environment all depend on the interaction between DNA and a CNT. 10 Individual DNA bases also bind to a graphitic surface through non-covalent π-π interactions. [11][12][13][14] Very little is known quantitatively about the strength of binding between ssDNA and CNT's in spite of the fundamental importance of understanding such interactions. 8,[15][16][17] As a first