The sequences required for stringent regulation of the E. coli tyrT gene have been analyzed in vivo. Stringent control was analyzed by nuclease-mapping RNA pulse-labeled with 32PO4. A 96 bp DNA fragment carrying the tyrT promoter was sufficient to confer regulation on a plasmid-encoded tyrT-galK fusion transcript. Deletion mutations that remove sequences upstream of the primary promoter elements greatly reduce promoter activity but do not remove the regulatory response. However, a 4 bp substitution mutation, adjacent to the transcription initiation site, disrupts stringent control. Thus the optimal expression and stringent regulation of the tyrT gene appears to result from the action of genetically separable promoter elements.