Escherichia coli RNA polymerase is a metalloenzyme containing 2 g-atoms of tightly bound zinc per mol of enzyme. We have prepared RNA polymerase from E. coli cells grown in a zinc-depleted medium supplemented with cobalt(II) chloride. The purified enzyme contains 1.8-~2.2 g-atoms of cobalt per mol of enzyme with concomitant reduction in the zinc content. The cobalt-substituted enzyme is enzymatically as active as Zn-RNA polymerase on a variety of templates under standard assay conditions. These two enzymes are almost identical by such physical criteria as subunit composition, monomer-dimer equilibrium, and pH and temperature stabilities. They differ in that Co-RNA polymerase exhibits a visible absorption spectrum with two major peaks at 584 and 703 nm. Addition of nucleoside triphosphates selectively perturbs the 584-nm peak, whereas addition of a template analogue, d(pT)10, affects both peaks. These spectral changes suggest that the tightly bound metal ions may directly or indirectly participate in binding of substrate or template to the enzyme. Biochemically, both enzymes are also very similar with respect to pH-activity profile, extrinsic metal requirements, 1, 10-phenanthroline inhibition, and fidelity of transcription of synthetic templates. Detailed kinetic and biochemical analyses have revealed that the Co enzyme has a lower value (~twofold) of apparent Km for T7 DNA under certain experimental conditions and that it is less efficient in initiating RNA chains at the A2 than at the A1 + A3 promoters on T7 DNA template as compared to the Zn enzyme. This has been demonstrated by studying the ratio of GTP/ATP incorporations into the 5′ terminal of RNA products and by measuring the formation of (rI)n-resistant initiation complexes at specific promoter sites using various combinations of dinucleotides and nucleoside triphosphates. Moreover, the in vitro transcription of a lac operon system by Co-RNA polymerase is less sensitive to cAMP and cAMP receptor protein than is the transcription by Zn-RNA polymerase. The results of our comparative studies using the Co and Zn enzymes further support the contention that the intrinsic metal of RNA polymerase is involved in promoter recognition and specific initiation in RNA synthesis.