This work investigates the effect on transcription of superhelical writhe located in the region immediately upstream of the -35 consensus sequence of Escherichia coli promoters. A set of double-stranded oligonucleotides, each with an unique DNA configuration, were designed, synthesized and substituted into an area of naturally occurring right-handed superhelical curvature immediately upstream of the beta-lactamase promoter in plasmid pUC19. All the mutants showed reduced promoter activities in E. coli cells. However, rightward superhelical writhe clearly facilitated transcription when compared with the effect produced by a straight DNA segment. Leftward writhe greatly repressed transcription. A plane curve showed an intermediate effect. This phenomenon was due not only to the difference in the ability of the segment to drive an open complex formation, but also to the difference in the binding affinity of RNA polymerase to the promoter. The positive effect of rightward writhe was also observed in vivo for the promoter of the tetracycline resistance gene of pBR322. The sense and extent of superhelical writhe of a DNA curvature seem to determine its influence on promoter activity.