Allostery in lac repressor ligand binding is based upon the ability of the protein to assume alternate ligand-bound conformations. The X-ray crystal structures of repressor bound to different ligands indicate the end points of conformational transitions. To detect changes in the environment within the repressor upon ligand binding, single tryptophan substitutions were generated in the following locations: the N-terminus (Tyr 7); the junction between the N-terminus and the N-subdomain (Leu 62); the N-subdomain of the monomer-monomer interface (Glu 100 and Gln117); the central region of the core (His 74, Tyr273, and Phe293); the C-subdomain (Phe226); and the C-terminus (Lys325). Changes in fluorescence properties upon inducer binding are only detected for tryptophans substituted at His74, Tyr273, and Phe 293. Fluorescence properties are not altered upon operator binding. Thus, in the regions of lac repressor probed by these substitutions, the inducer-bound form differs from the conformation of the unliganded form. The roles of His74 and Asp278 in operator and inducer binding were also explored. His74 and Asp 278 form a potential salt bridge between the N- and C-subdomains in the repressor core. Mutations were generated at both positions to vary size, charge, and polarity in an effort to determine the nature and importance of this interaction. All repressor proteins produced by mutating His74 and/or Asp278 possess altered ligand binding behavior. Interestingly, only the H74W mutant repressor did not display the characteristic ∼1000-fold decrease in operator affinity when bound to inducer. The oligomeric state of lac repressor is a result of the free energy involved in both assembly and folding. The overall stability of tetrameric repressor was determined by two approaches. The free energy of complete unfolding by urea-induced denaturation of tetrameric repressor is ∼49.1 kcal/mol. The total free energy for denaturation of tetrameric repressor calculated by combining all of the assembly and folding events is ∼58.8 kcal/mol. These results demonstrate unequivocally that the tetrameric lac repressor is an extremely stable protein. However, the difference between these free energy values suggests that the same transitions may not be monitored in the two different approaches.