The DNA-dependent RNA polymerase from Escherichia coli possesses 2 g-atoms of tightly bound Zn ions per mol of holoenzyme (αββ′σ). These two Zn ions are in the core enzyme (αββ′), and the isolated a subunit is essentially free of Zn. None of the individual subunits (α,β,β or ′) isolated from phosphocellulose chromatography of core enzyme contains significant amounts of Zn in the presence of 7 M urea, indicating that the intrinsic Zn ions have been released from core enzyme by denaturation. Similar results were also observed for the α′ subunit and α2β complex separated from urea-denatured core polymerase by Affi-Gel Blue chromatography. However, when these isolated subunits or subunit complex were dialyzed against buffer containing 10-5 M Zn ions to remove urea followed by dialysis against 10 mM EDTA to remove free and loosely bound Zn ions, we found that the isolated β and β′ subunits contain 0.6 ± 0.3 and 1.4 α 0.5 gatoms of tightly bound Zn ions per mol of subunit, respectively The isolated a subunit has less than 0.1 g-atom of Zn per mol of subunit. This finding together with the fact that the isolated subunits can be reconstituted into active enzyme strongly suggest that at least one of the two tightly bound Zn ions in RNA polymerase is located in β′ subunit, while the other Zn ion may be in β′, or β, or at the contact domain of these two subunits. This conclusion is further supported by studies with Co-RNA polymerase in which the two intrinsic Zn ions have been replaced by Co(II) ions. Oxidation by H2O2 transforms Co(II) to the exchange-inert Co(III) state and, therefore, selectively “freezes” Co ions in their respective binding sites. It was found that at least one Co(IIl) ion is bound tightly to the isolated β′ subunit even in the presence of 6.5 M urea. The subunit locations of the two tightly bound Zn ions in RNA polymerase imply that these intrinsic metal ions may play both functional and structural roles in gene transcription.