The present work reveals the entropic preference in the two-step binding process of small molecule DNA minor groove binders (MGBs), involving the formation of a dimer in free solution followed by the binding of that dimer to DNA, which contrasts with the sequential or simultaneous cooperative binding of two unbound MGB molecules with DNA that is implicitly assumed in the majority of reported DNA-MGB binding studies. The results are generalized for the n-mer binding case and the methodological outcomes are discussed. (C) 2015 Elsevier B.V. All rights reserved.