This is the first report to describe the detailed analysis of TATA sequence requirements for the initiation of transcription for RNA polymerase II in a plant in vitro transcription system. We examined the transcriptional activities associated with 24 promoters, including wild-type (TATATATA), and the 21 point and two multiple mutations in tobacco, Drosophila, and HeLa in vitro systems. Although the relative transcriptional activities varied among these promoters, all three systems gave similar results. These results suggest the functional conservation of TATA sequence requirements for in vitro transcription systems among plants, insects, and human