Work from a number of laboratories has indicated that the TATA box sequence can act as a basal promoter element not only for RNA polymerase II (RNAP II) transcription, but also for transcription by RNA polymerase III (RNAP III). We previously reported that, in the absence of other cis-acting elements, the canonical TATA sequence TATAAAAA specifically supported transcription by RNAP II in an unfractionated Drosophila nuclear extract, whereas the sequence TTTTTATA (the same sequence in reverse orientation) directed RNAP III transcription. We have now examined a variety of other TATA box sequences with regard to RNA polymerase selectivity and their ability to support RNAP III transcription. The results have allowed us to rank these TATA box sequences with respect to their relative strengths as RNAP III promoter elements in unfractionated Drosophila extracts. Further, the data indicate that T residues at positions 2 and 4 of the TATA box appear to be important determinants of RNAP III selectivity in this system, whereas A residues at these positions favor RNAP II transcription. Finally, the data suggest that transcription factors TFIID and TFIIIB, although both capable of binding a variety of TATA elements, have distinct sequence preferences for recognizing the TATA box and possibly the surrounding DNA.