In order for RNA polymerase (pol) III to produce a sufficient quantity of RNAs of appropriate structure, initiation, termination, and reinitiation must be accurate and efficient. Termination-associated factors have been shown to facilitate reinitiation and regulate transcription in some species. Suppressor tRNA genes that differ in the dT(n) termination signal were examined for function in Schizosaccharomyces pombe. We also developed an S. pombe extract that is active for tRNA transcription that is described here for the first time. The ability of this tRNA gene to be transcribed in extracts from different species allowed us to compare termination in three model systems. Although human pol III terminates efficiently at 4 dTs and S. pombe at 5 dTs, Saccharomyces cerevisiae pol III requires 6 dTs to direct comparable but lower termination efficiency and also appears qualitatively distinct. Interestingly, this pattern of sensitivity to a minimal dT(n) termination signal was found to correlate with the sensitivity to alpha-amanitin, as S. pombe was intermediate between human and S. cerevisiae pols III. The results establish that the pols III of S. cerevisiae, S. pombe, and human exhibit distinctive properties and that termination occurs in S. pombe in a manner that is functionally more similar to human than is S. cerevisiae.