Homeodomains are helix-turn-helix type DNA-binding domains that exhibit sequence-specific DNA binding by insertion of their “recognition” R helices into the major groove and a short N-terminal arm into the adjacent minor groove without inducing substantial distortion of the DNA. The stability and DNA binding of four representatives of this family, MATR2, engrailed, Antennapedia, and NK-2, and truncated forms of the last two lacking their N-terminal arms have been studied by a combination of optical and microcalorimetric methods at different temperatures and salt concentrations. It was found that the stability of the free homeodomains in solution is rather low and, surprisingly, is reduced by the presence of the N-terminal arm for the Antennapedia and NK-2 domains. Their stabilities depend significantly upon the presence of salt: strongly for NaCl but less so for NaF, demonstrating specific interactions with chloride ions. The enthalpies of association of the homeodomains with their cognate DNAs are negative, at 20 °C varying only between -12 and -26 kJ/mol for the intact homeodomains, and the entropies of association are positive; i.e., DNA binding is both enthalpy- and entropy-driven. Analysis of the salt dependence of the association constants showed that the electrostatic component of the Gibbs energy of association resulting from the entropy of mixing of released ions dominates the binding, being about twice the magnitude of the nonelectrostatic component that results from dehydration of the protein/DNA interface, van der Waals interactions, and hydrogen bonding. A comparison of the effects of NaCl/KCl with NaF showed that homeodomain binding results in a release not only of cations from the DNA phosphates but also of chloride ions specifically associated with the proteins. The binding of the basic N-terminal arms in the minor groove is entirely enthalpic with a negative heat capacity effect, i.e., is due to sequence-specific formation of hydrogen bonds and hydrophobic interactions rather than electrostatic contacts with the DNA phosphates.