Abstract In a transcription system containing λpgal8 DNA as template and Escherichia coli RNA polymerase, the addition of cyclic adenosine monophosphate receptor protein (CRP) and 3',5'-cyclic AMP causes a 15-fold increase in gal mRNA synthesis. Gal mRNA was measured by hybridizing the [3H]RNA product to the separated strands of λpgal8 DNA after removal of λ mRNA by preliminary hybridization to λ DNA. Competition experiments indicate that a large fraction of this RNA is identical with in vivo gal mRNA. With λpgal8 DNA as template but not with λ DNA, 3',5'-cyclic AMP and CRP cause a 2-fold increase in λ mRNA. Since the increment in λ mRNA hybridizes to the left-hand half of the ι strand of λ DNA and is abolished by the addition of ρ, it is interpreted as read through from the 3',5'-cyclic AMP-CRP sensitive site in the gal region of λpgal8 DNA. The concentrations of CRP and 3',5'-cyclic AMP necessary for half-maximal stimulation of gal mRNA synthesis are 5 x 10-8 and 5 x 10-6 m, respectively. 2',3'-Cyclic AMP and 5'-AMP do not stimulate gal mRNA synthesis; 3',5'-cyclic GMP is an inhibitor. When 3',5' cyclic AMP, CRP, DNA, and RNA polymerase are incubated together for 10 min, a rifampicin-resistant preinitiation complex is formed, and, upon the addition of nucleoside triphosphates, gal mRNA synthesis occurs. 3',5'-Cyclic GMP prevents the formation of this complex and can dissociate it after it has been formed. These results suggest that 3',5'-cyclic AMP and CRP stimulate gal transcription at a step prior to chain elongation.