The TATA box is a key upstream control element for basal tRNA gene transcription by RNA polymerase III in some eukaryotes, such as the fission yeast (Schizosaccharomyces pombe) and higher plants, but not in others such as the budding yeast (Saccharomyces cerevisiae). To gain information on this differential TATA box requirement, we examined side-by-side the in vitro transcription properties of TATA-containing and TATA-mutated plant and S. cerevisiae tDNAs in homologous in vitro transcription systems from both organisms and in a hybrid system in which yeast TBP was replaced by its plant homologue. The data support the general conclusion that specific features of the plant transcription machinery, rather than upstream region architecture per se, are responsible for the much stronger TATA box dependence of the plant system. In both systems, however, a strong influence of the TATA box on transcription start site selection was observed. This was particularly striking in the case of plant tDNAs, where TATA-rich upstream regions were found to favour the use of alternative initiation sites. Replacement of yeast TBP with its plant counterpart did not confer any general TATA box responsiveness to the yeast transcription machinery. Interactions involving components other than TBP are thus responsible for the strong TATA box requirement of plant tDNA transcription.