Trp repressor regulates the expression of TrpEDCBA, aroH, and TrpR operons. Recently, X-ray crystallographic analysis of a Trp repressor-operator complex has suggested a stoichiometry of one repressor dimer to one operator without any direct hydrogen bonds between repressor residues and operator base pairs in the major groove, a situation termed "indirect readout" (Otwinowski et al., 1988). In this study, we have systematically characterized the repressor-operator interactions using repressor mutants and synthetic operators. Trp repressor binds to TrpEDCBA and aroH, but not to TrpR, 40 bp operator DNAs with the same stoichiometry and with similarly binding affinity. The direct stoichiometry measurements with a symmetric variant of TrpEDCBA operator DNA show the stoichiometry to be two repressor dimers to one operator. In addition, Trp repressor binds to a hybrid 40 bp DNA containing half of the TrpEDCBA half binding site and half non-specific DNA with reduced affinity but with the same apparent stoichiometry based on relative gel mobility, that is, two repressor dimers even to one half binding site operator. Although this stoichiometry is different from those proposed in the literature, it is consistent with our observations that (1) there is an unusually large interface in the Trp repressor-operator interaction, (2) Trp repressor forms tetramer in solution, and (3) in the repressor-operator interaction there are two binding reactions, where some cooperativity may be involved. It is further observed that repressor binding reactions can be altered by mutations in either operator or repressor. The higher stoichiometries observed for superrepressors may reflect the molecular basis for the enhanced stability of superrepressor-operator complex. Furthermore, data derived from studies on supperrepressors suggest that the repressor amino terminus could be involved in the dimer-dimer interaction that may occur in the formation of repressor-operator complex. UV footprinting analyses of bromodeoxyuridine-substituted Trp operator DNA show that the binding environments in the operator sequence are asymmetric and that some unique features of the Trp repressor-operator interaction are also evident. Finally, Trp repressor appears to have no close contact with operator DNA in the major groove near the 5-position of thymidine residues or it has no reactive amino acid residues in the vicinity of the 5-position of the thymidine, as no crosslinked repressor-operator complex was found in our study.