High-density monolayers (HDMs) of single-strand (ss) DNA are important nanoscale platforms for the fabrication of sensors and for mechanistic studies of enzymes on surfaces. Such systems can be used, for example, to monitor gene expression, and for the construction of more complex nanodevices via selective hybridization with the complementary oligos dissolved in solution. In this framework, controlling HDM hybridization is essential to control the final properties. Different studies demonstrate that at the typical density of approximate to 10(13) molecules per cm(2) no more than approximate to 30-40% of the HDM ssDNA is successfully hybridized. Until now, however, the origin of the HDM hybridization limit has remained unclear. In this work, molecular dynamics (MD) simulations of HDM systems with variable hybridization reveal that, independently of other experimental parameters, the effective hybridization for a HDM of this density is intrinsically limited by molecular and electrostatic crowding. A detailed structural analysis of the HDM model shows good agreement with our atomic force microscopy (AFM) experiments, and provides further insight into the steric hindrance behaviour and time-resolved surface topography of these nanostructured systems. The explicit relationship proposed between structural crowding and limited HDM hybridization offers a rationale to control the final properties of HDM-based nanodevices.