The inducer binding site of the E. coli lactose repressor has been examined by in vitro characterization of mutant proteins with amino acid substitutions at the hypothetical inducer binding site (Sams, C. F., Vyas, N. K., Quiocho, F. A. and Matthews, K. S. (1984) Nature 310, 429-430). Since the major force for sugar interaction comes from hydrogen bonding between the inducer and the hydrophilic side chains in the inducer binding site of lac repressor, Lys\sp84, Asp\sp88, Asp\sp130 and Asp\sp274 at the hypothetical inducer binding site were chosen to be substituted. Using site-directed mutagenesis, each site was substituted with four different amino acids that altered side chain length, polarity or charge. In addition, double mutations were introduced at Lys\sp84 and Tyr\sp282 sites in order to elucidate the role of Lys\sp84 in subunit interaction. Through characterizing mutant proteins at the Lys\sp84 site in vitro, including inducer binding properties, operator binding activities, immunoblotting pattern, and gel filtration behavior, we conclude that Lys\sp84 is at the same subunit interface as Tyr\sp282 and is not involved directly in the inducer binding site. A similar situation applies to Asp\sp88, which does not appear to be in the inducer binding site and does not directly contribute energy to inducer binding. The data on mutants at Asp\sp130 indicate no effect on inducer binding, while mutations at Asp\sp274 suggest an essential role is played by this amino acid. Based on the fact that mutations at the Asp\sp274 site did not show any spectroscopic alteration in the presence of inducer, even though the presence of IPTG protected iodide quenching of Trp\sp220 in the inducer binding site, we propose that this amino acid is essential for the conformational change upon inducer binding. This proposal is strongly supported by the X-ray crystallographic data obtained for periplasmic sugar binding proteins whose sequences show homology to lac repressor core domain. Thus, all the mutational data presented in this study are consistent with the recently proposed structure of lac core protein which is based on sequence alignment and molecular replacement modeling (Nichols, J. C., Vyas, N. K., Quiocho, F. A. and Matthews, K. S. (1993) J. Biol. Chem. 268, 17602-17612).