The equilibrium binding of several anilinoacridine analogs are compared over a wide range of ionic strengths and temperatures. Although o-AMSA binds DNA with a higher affinity than m-AMSA it is not effective as an antitumor agent. Both m-AMSA and o-AMSA bind DNA in an intercalative manner. In an effort to gain insight into the physical chemical properties associated with these compounds and to correlate these properties with antitumor activity, an in-depth investigation into the thermodynamic parameters of these compounds and structurally related anilinoacridine analogs was performed. These studies demonstrate that substituient type and placement on the aniline ring of the anilinoacridines influence both the affinity of these drugs in binding to DNA and dictate whether the DNA binding is an enthalpy or entropy driven process. These data demonstrate that the antitumor agent m-AMSA interacts with DNA via an enthalpy driven process. In contrast, the structurally similar but biologically inactive o-AMSA binds to DNA through an apparent entropy driven process. The differences in thermodynamic mechanisms of binding between the two isomers along with molecular modeling studies reveal that the electronic and/or steric factors resulting from the positioning of the methoxy substituient group on the anilino ring directs the DNA binding properties through orientation of the methanesulfonamido group at the l’ position of the aniline ring. The orientation of this substituient group may result in favorable contacts through hydrogen bonding with neighboring base-pairs and ultimately influence the biological effectiveness as an antitumor agent.