Threonine is found at the third position of the second alpha-helix in the helix-turn-helix motifs of most bacterial DNA-binding proteins. To investigate the role of this conserved residue in Escherichia coli Trp repressor function, plasmids encoding mutant Trp repressors with each of the 19 amino acid changes of Thr-81 were made by site-directed mutagenesis. All 19 changes decrease the activity of Trp holorepressor, indicating that the Thr-81 side-chain is critical for TrpR function. Three mutant repressors, Ser-81, Lys-81 and Arg-81, retain partial DNA-binding activity and inhibit transcription from the wild-type trp promoter/operator complex; challenge-phage assays show that Ser-81 and Lys-81 holorepressors have altered DNA-binding specificities. The side-chain of Thr-81 may make direct contacts with base pairs 4 and 3 of the trp operator, consistent with the nuclear magnetic resonance solution structures of the holorepressor-operator complex.