A plasmid carrying a CRP-dependent promoter fused to the lac structural genes was manipulated to construct a set of spacing mutants that have varying lengths between the CRP binding site and the -35 region. The lengths of the spacer were changed over 45 bp by inserting or deleting nucleotides. DNase I footprinting analysis revealed that the spacer length did not affect the binding of cAMP-CRP to the CRP site. The effect of the spacer length on transcription activation by cAMP-CRP was tested in vivo by beta-galactosidase and quantitative S1 assays with crp+ and delta crp cells harboring plasmids. Insertions or deletions of non-integral helical turns, which displace the CRP site onto the opposite face of DNA helix compared to the original promoter, eliminated completely the activation of transcription. In contrast, changing the spacer length by integral helical turns allowed the promoter to respond to CRP, although the degree of activation varied with the length of the spacer. We conclude that stereospecific positioning of CRP and RNA polymerase on the DNA helix is strictly required for CRP action. The data support a model that CRP stimulates transcription by directly contacting RNA polymerase.