With the aim to gain a better understanding of the various driving forces that govern sequence specific DNA minor groove binding, we performed a thermodynamic analysis of netropsin binding to an AT-containing and to a set of six mixed AT/GC-containing binding sequences in the DNA minor groove The relative binding free energies obtained using molecular dynamics simulations and free energy calculations show significant variations with the binding sequence. While the introduction of a GC base pair in the middle or close to the middle of the binding site is unfavorable for netropsin binding, a GC base pail at the end of the binding site appears to have no negative influence on the binding The results of the structural and energetic analyses of the netropsin DNA complexes reveal that the differences in the calculated binding affinities cannot be explained solely in terms of netropsin DNA hydrogen-bonding or interaction energies In addition. solvation effects and entropic contributions to the relative binding free energy provide a more complete picture of the various factors determining binding. Analysis of the relative binding entropy indicates that its magnitude is highly sequence-dependent, with the ratio vertical bar T Delta Delta S vertical bar/vertical bar Delta Delta H vertical bar ranging from 0.07 for the AAAGA to 1.7 for the AAGAG binding sequence, respectively