Analysis of the termination of transcription by yeast RNA polymerase I (Pol I) using in vitro run-on experiments in both isolated nuclei and permeabilized cells demonstrated that Pol I does not traverse the whole intergenic spacer separating consecutive 37S operons, but terminates transcription before reaching the 5S rRNA gene, that is within NTS 1. In order to discriminate between processing and termination at the 3'-end generating sites previously identified in vivo in NTS 1 (T1, T2 and T3), fragments containing these sites were inserted into the middle of the reporter DNA of an artificial rRNA minigene. RNA isolated from yeast cells transformed with these minigenes was analyzed for the presence of transcripts derived from sequences both up- and downstream of the insert by Northern blot hybridization, reverse transcription analysis and S1 nuclease mapping. In accordance with previously obtained results T1 (+15 to +50) was found to behave as a processing site. T2 (+210) however was concluded to be an efficient, genuine Pol I terminator. In addition to T2, two other terminators were identified in NTS 1: T3A (at +690) and T3B (at +950). Surprisingly, when the 3' terminal part of NTS 2 was tested for its capacity to generate 3'-ends, another terminator (Tp) was found to be present at a position 300 bp upstream of the transcription initiation site of the 37S-rRNA operon.