Knowledge of the sequence of the human genome has provided significant opportunities to exploit DNA as a target in the rational design of therapeutic agents. Among agents that target DNA, netropsin exhibits a strong preference for binding A/T rich regions. In order to investigate the key factors responsible for DNA recognition and binding by netropsin, molecular dynamics simulations were carried out on a DNA-netropsin complex in which two netropsin molecules are bound to each AATT site of the 16-mer d(CTTAATTCGAATTAAG)(2). In this complex, the two netropsins are bound to the DNA minor groove in a head-to-head orientation with the guanidinium-termini of both netropsins pointed toward the center of the DNA. Despite their identical environments, molecular dynamics simulations showed that the two netropsins exhibited differences in their respective RMS behaviors, binding energies, minor groove width fluctuations, and rotations of their structural planes. These observations suggest that DNA recognition and binding by small molecules may be governed by mechanism(s) that are much more complex than initially anticipated and may represent unexpected challenges in genome-targeted drug design.