The three-dimensional structure of DNA-dependent RNA polymerase (RNAP) from thermophilic Thermus aquaticus has recently been determined at 3.3 A resolution. Currently, very little is known about T. aquaticus transcription and no genetic system to study T. aquaticus RNAP genes is available. To overcome these limitations, we cloned and overexpressed T. aquaticus RNAP genes in Escherichia coli. Overproduced T. aquaticus RNAP subunits assembled into functional RNAP in vitro and in vivo when coexpressed in E. coli. We used the recombinant T. aquaticus enzyme to demonstrate that transcription initiation, transcription termination, and transcription cleavage assays developed for E. coli RNAP can be adapted to study T. aquaticus transcription. However, T. aquaticus RNAP differs from the prototypical E. coli enzyme in several important ways: it terminates transcription less efficiently, has exceptionally high rate of intrinsic transcript cleavage, and is highly resistant to rifampin. Our results, together with the high-resolution structural information, should now allow a rational analysis of transcription mechanism by mutation.