The regulatory region of the tryptophan operon of Escherichia coli was subjected to in vitro site-directed mutagenesis using sodium bisulfite as the mutagen. The mutagenized DNA was used to transform cells to drug resistance, and plasmid DNAs from individual transformants were isolated and sequenced. Overall, 22% of the plasmids sequenced contained alterations within the region of interest. Many of the mutants obtained had characteristics that bear on aspects of the alternative secondary structure model of attenuation. Expression analyses with several of the mutants provided evidence suggesting that ribosome dissociation at the leader peptide stop codon may be rapid and that this dissociation is responsible for setting the steady-state level of expression observed in cultures growing in the presence of excess tryptophan. One mutation altered the amino acid composition of the leader peptide without affecting a transcript secondary structure. The behavior of this mutant supports the prediction that the leader peptide per se plays no role in attenuation, rather it is the act of synthesis of the peptide that has regulatory significance. Several of the mutations provide information on the structure of the RNA antiterminator . Additional mutations support the conclusion that the last stem and loop structure in the terminated transcript, structure 3:4, is sufficient to cause transcription termination.