A crude in vitro transcription system which selectively transcribes DNA fragments containing the promoter region of the Tetrahymena pyriformis rRNA gene has been prepared from T. pyriformis. The system requires both an S100 fraction of lysed isolated macronuclei and an S100 extract of whole cells. When a HhaI-HindIII fragment of the promoter containing plasmid pEN 19-1 is employed as a template, transcription yields two major products of about 560 (A) and 510 (B) bases in length. The analysis of the transcription products of truncated templates showed that RNA A is a runoff transcript and RNA B is produced by nucleolytic cleavage of RNA A at a site about 50 nucleotides to the left of the HindIII cleavage site. S1 nuclease mapping was used to demonstrate that the 5' end of RNA A is identical with that predicted for a transcript which was initiated at the same site on the gene as the in vivo 35S rRNA precursor. Transcription is dependent upon the addition of promoter containing DNA, is inhibited by 1 microgram/mL actinomycin D, and is insensitive to 200 micrograms/mL alpha-amanitin. Transcription is dependent upon the salt levels in the assay exhibiting activity peaks at 58 mM KCl, 28 mM (NH4)2SO4, and 3 mM MgCl2. Several minor transcription start sites to the left of the major initiation site become active at high salt, yielding several minor longer transcripts. High salt also inhibits the RNA cleavage activity, reducing the levels of RNA B produced.