Transcription with fission yeast (Schizosaccharomyces pombe) RNA polymerase III (pol III) was studied in two different in vitro systems. Reactions performed with isolated nuclei gave rise to 5S and pre-tRNA molecules. Because the alpha-amanitin sensitivity of that reaction clearly differed from what has been observed with pol III enzymes of other eukaryotes, a cell-free S. pombe transcription extract was developed and analyzed with the homologous 7S L RNA (srp RNA; signal recognition particle RNA) gene. Synthesis of 7S L RNA was found to be sensitive to high concentrations of alpha-amanitin, with 50% reduction seen at 400 microg/ml of the toxin. However, even with very high alpha-amanitin concentrations, exceeding 1 mg/ml, no full inhibition of the S. pombe pol III enzyme could be obtained. Together, these results demonstrate that in contrast to the yeast Saccharomyces cerevisiae, pol III from S. pombe is sensitive to high concentrations of alpha-amanitin, yet with a clearly different dose response than that observed with the corresponding RNA polymerase of higher eukaryotes. Furthermore, while the S. pombe 7S L RNA gene was efficiently transcribed in HeLa cell extracts, the human 7S L RNA gene was not actively transcribed in the S. pombe system. This finding of divergent promoter structures of both genes was verified by the analysis of 5' deletion mutants of the S. pombe 7S L RNA gene.