Abstract: RNA polymerase (Pol) III, assisted by TFIIIC and TFIIIB, transcribes small untranslated RNAs, such as tRNAs. In addition to known Pol III-transcribed genes, the Saccharomyces cerevisiae genome contains loci (ZOD1, ETC1-8) associated to incomplete Pol III transcription complexes (Moqtaderi and Struhl, K. 2004, Mol. Cell. Biol. 24, 4118-4127). We show that a short segment of the ZOD1 locus, containing box A and box B promoter elements and a termination signal between them, directs the Pol III-dependent production of a small RNA both in vitro and in vivo. In yeast cells, the levels of both ZOD1- and ETC5-specific transcripts were dramatically enhanced upon nucleosome depletion. Remarkably, transcription factor and Pol III occupancy at the corresponding loci did not change significantly upon derepression, thus suggesting that chromatin opening activates poised Pol III to transcription. Comparative genomic analysis revealed that the ZOD1 promoter is the only surviving portion of a tDNAIle ancestor, whose transcription capacity has been preserved throughout evolution independently from the encoded RNA product. Similarly, another TFIIIC/TFIIIB-associated locus, close to the YGR033c ORF, was found to be the strictly conserved remnant of an ancient tDNAArg. The maintenance, by eukaryotic genomes, of chromatin-repressed, non-coding transcription units has implications for both genome expression and organization.