The cI repressor of bacteriophage 434, known as 434 repressor, binds to 14-bp operator sequences by means of a helix-turn-helix motif. To probe the requirements for selective DNA recognition by this class of DNA binding proteins, as well as to generate new proteins with altered specificities, a library of approximately 3 x 10(6) mutants was generated that contains all permutations of five residues in the recognition helix (helix 3) of the repressor. These mutants were then selected in vivo for their ability to bind both wild-type (WT) and mutant operator sequences. The results of the selection demonstrate that four of these residues--Gln28, Gln29, Ser30, and Gln33--play a critical role in recognition of the WT operator. A number of repressors with mutations at Thr27 showed altered DNA binding affinities and specificities. The approach described here may also prove useful in studies of DNA recognition by other classes of DNA binding proteins.