RNA polymerase holoenzyme from Thermus thermophilus, consisting of six protein subunits (α2, β, β′, ω and σ70) and having a total molecular mass of about 450 kDa, was purified and crystallized by the hanging-drop vapour-diffusion technique under mild near-physiological conditions. The crystals diffract beyond 3 Å resolution. Careful analysis of diffraction data revealed that the crystals belong to space group P32, with unit-cell parameters a = b = 236.35, c = 249.04 Å, and have perfect twinning along the threefold axis. A complete data set at 3 Å resolution was collected and an unambiguous molecular-replacement solution was found using the structure of T. aquaticus RNA polymerase core enzyme as a search model. The refinement of structure and model building of the σ70 subunit is now in progress.