DNA-dependent RNA polymerase was purified from Pseudomonas aeruginosa. The subunit structure was typical of other eubacterial RNA polymerases in having β′ (157 000), β (148 000), σ (87 000), and α₂ (45 000) subunits as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme was dependent on Mg²⁺, displaying optimal activity at 10 mM MgCl₂. Ca²⁺ and Zn²⁺ could not replace MgCl₂ in the assay system, while Mn²⁺ produced partial activity. KCl at concentrations greater than 10 mM inhibited enzyme activity. Optimal enzyme activity was observed at pH 8.5–9.0. The RNA polymerase was stable in 50% (w/v) glycerol at 4 °C for more than 3 months. Enzyme activity was inhibited in vitro by heparin, streptolydigin, streptovaracin, actinomycin D, and rifampicin.