Structural behaviors of cytosine in the hydrated Na+- montmorillonite clay interlayer have been investigated via ab initio molecular dynamics simulations. Short constrained simulations have been carried out by increasing the distance between the nucleobase and the surface. Structural data shows that a three water layer structure is more or less maintained in the interlayer, depending on the orientation of the nucleobase. Close to the surface, the cytosine remains in a coplanar orientation, which is stabilized by dispersion forces between the π system of cytosine and the surface. The Na+ cation is adsorbed on the surface and is coordinated to cytosine through the O2 heteroatom, more accessible than N3, the latter interacting with water. The water interlayer molecules interact with both the nucleobase heteroatoms and the adsorbed cation. However, when the nucleobase gets away from the surface, it adopts an almost orthogonal orientation and interacts with the surface oxygen atoms via hydrogen bonds between the C5 and the amino hydrogen atoms. The Na+ cation becomes hydrated and coordinates to cytosine via both the N3 and O2 atoms. The profile of the forces between cytosine and the surface helped us to identify stable configurations of cytosine at different nucleobase-clay distances. Longer simulations for the observed stable configurations have shown that the nucleobase remains stacked over the surface up to a distance of 4.9 Å. At a larger distance cytosine engages hydrogen bonds with the surface, in a more or less defined orthogonal orientation. © 2013 American Chemical Society.