Halorhodopsin from Natronomonas pharaonis (NpHR) acts an inward-directed, light-driven chloride pump and forms a homotrimer. To evaluate effect of trimeric assembly, that is, intermolecular interaction, on the control or modulation of light-driven chloride pumping activity of individual HRs, it is important to understand the thermal and chloride sensitivity of trimer dissociation and the structural stability of HR. In this study, the thermal dissociation of NpHR trimer to monomer in a dodecyl β-D-maltoside- solubilized system was investigated, using size-exclusion chromatography and visible absorption. In the absence of Cl-, NpHR retained the trimer assembly at 25 °C but dissociated to the monomer with an increase in temperature to > 40 °C. On the other hand, in the presence of Cl -, the trimer assembly was maintained at 40 °C. The dissociation of the trimer to the monomer after incubation at 40 °C, which was determined via sizeexclusion chromatography, depended on the Cl- concentration and showed a sigmoidal isotherm. From this isotherm, the apparent dissociation constant for Cl- was estimated to be 22mMwith a Hill coefficient of 2.2. A similar isotherm was obtained when SO42- was used instead of Cl- with a dissociation constant of 94 mM. On the other hand, thermal dissociation of the NpHR trimer to the monomer in the absence of Cl- proceeded by two components: the fast component is susceptible to the changes in temperature and detergent concentration, and the slow component is accompanied by bleaching at the same time. Activation energies of the fast and slow dissociation components and bleaching were 57.8, 35.3, and 40.5 kcal/mol, respectively. The presence of a second chloride-binding site with a Hill coefficient of ∼2 at the surface of NpHR to control the trimer-monomer conversion was discussed.