Abstract Expression of the Salmonella typhimurium pyrD gene was found to be repressed two-fold when cells were grown in the presence of hypoxanthine. Purine-mediated repression was evident for reporter plasmids containing pyrD-lacZ transcriptional or translational fusions, indicating that regulation was being exercised at the level of transcriptional initiation. In a strain harbouring a purR6::Tn10 mutation inactivating the purine regulon repressor (PurR), expression of pyrD was not repressed by hypoxanthine. Gel retardation experiments provided evidence that PurR binds to a PUR box centered 27 base pairs upstream of the upstream of the −35 region of the pyrD promoter. Site-directed mutagenesis was used to decrease the similarity of the putative PUR box to the consensus sequence; each mutation eliminated binding of PurR to the mutated DNA in vitro and abolished repression by hypoxanthine in purR+ cells in vivo. Regulation by pyrimidines was unaffected by either of the two PUR box mutations, showing that purine and pyrimidine control of pyrD expression can operate independently.