CysB is a transcriptional activator for the cysteine regulon and negatively autoregulates its own gene, cysB. Transcription activation also requires an inducer, N-acetyl-L-serine. CysB is known to bind to activation sites just upstream of the -35 regions of the positively regulated cysJIH, cysK, and cysP promoters and to a repressor site centered at about +1 in the cysB promoter. Additional accessory sites have been found in positively regulated promoters. The hydroxyl radical footprinting experiments reported here indicate that the activation sites CBS-J1, CBS-K1, and CBS-P1 in the cysJIH, cysK, and cysP promoters are composed of two convergently oriented 19-bp half-sites separated by 1 or 2 bp. N-Acetyl-L-serine stimulates binding to these sites as well as to the accessory sites CBS-J2 and CBS-P2, both of which share a similar topology with activation sites. A second topology is found in the accessory site CBS-K2 and the repressor site CBS-B, which contain divergently oriented 19-bp half-sites separated by one or two helical turns. N-Acetyl-L-serine inhibits binding to these two sites. A third topology is present in the cysK and cysP promoters, where an additional half-site is oriented toward the activation site and separated from it by one helical turn. Here, CysB binds to all three half-sites, bending the DNA, and N-acetyl-L-serine decreases the extent of bending. The marked dissimilarities of these half-site arrangements and of their responses to N-acetyl-L-serine suggest that CysB, a homotetramer, binds to them with different combinations of subunits.