The interaction of the Salmonella typhimurium virulence gene regulator, SpvR, with its operator sites upstream of the spvA and spvR genes was analysed in vivo by dimethyl sulphate (DMS) footprinting and site-directed mutagenesis. DMS methylation protection assays showed that, in vivo, SpvR forms direct protein-DNA contacts with nucleotides clustered in two regions (+1 to −27 and −51 to −71) of the spvA regulatory region. These regions were subjected to site-directed mutagenesis and the effects on SpvR binding and gene activation assessed. Mutations that prevented occupancy of the promoter distal site (−51 to −71) in vivo also prevented occupancy of the promoter proximal site (+1 to −27), whereas mutations in the proximal site affected binding only at the proximal site and not the distal site. SpvR binding at the promoter proximal site was an essential prerequisite for transcription activation. These findings demonstrated a hierarchy of SpvR binding in which the promoter distal site is dominant to the proximal. The spvR gene was found to possess an operator site that resembled closely the distal SpvR binding site of the spvA operator. Nonetheless, SpvR interaction with the spvR operator was difficult to detect in vivo. When the nucleotide sequence of the spvR operator was altered at two nucleotides so that it corresponded more precisely to that of the distal site of the spvA operator, strong SpvR-DNA interactions were detected, with nucleotides in the region −31 to −67 being protected from DMS methylation in vivo. However, despite the improved interaction with the transcriptional activator, the altered regulatory region was poorer at promoting spvR gene transcription than the wild type. We describe a two-step model for activation of the spvA promoter and discuss the possibility that a specific cofactor in addition to sigma factor RpoS is required for SpvR action at this promoter in vivo.