Abstract: Eukaryotic RNA polymerase (Pol) III terminates transcription at short runs of T residues in the coding DNA strand. By genomic analysis, we found that T5 and T4 are the shortest Pol III termination signals in yeasts and mammals, respectively, and that, at variance with yeast, oligo(dT) terminators longer than T5 are very rare in mammals. In Saccharomyces cerevisiae, the strength of T5 as a terminator was found to be largely influenced by both the upstream and the downstream sequence context. In particular, the CT sequence, which is naturally present downstream of T5 in the 3' flank of some tDNAs, was found to act as a terminator-weakening element that facilitates translocation by reducing Pol III pausing at T5. In contrast, tDNA transcription termination was highly efficient when T5 was followed by an A or G residue. Surprisingly, however, when a termination-proficient T5 signal was taken out from the tDNA context and placed downstream of a fragment of the SCR1 gene, its termination activity was compromised, both in vitro and in vivo. Even the T6 sequence, acting as a strong terminator in tRNA gene contexts, was unexpectedly weak within the SNR52 transcription unit, where it naturally occurs. The observed sequence context effects reflect intrinsic recognition properties of Pol III, because they were still observed in a simplified in vitro transcription system only consisting of purified RNA polymerase and template DNA. Our findings strengthen the notion that termination signal recognition by Pol III is influenced in a complex way by the region surrounding the T cluster, and suggest that readthrough transcription beyond T clusters might play a significant role in the biogenesis of class III gene products.