TGA1a is a tobacco DNA-binding protein that binds to the activation sequence-1 (as-1) element of the cauliflower mosaic virus 35S promoter. We have produced TGA1a in Escherichia coli, purified it from bacterial extracts, and examined its effect on transcription in a human in vitro system. Addition of TGA1a stimulates transcription by up to 20 times, and the stimulation is dependent on the presence of the as-1 element in the promoter. When transcription reinitiation is inhibited by 0.3 M KCl, activation is similar. Therefore, TGA1a activates transcription by increasing the number of active preinitiation complexes. After formation of the preinitiation complexes in the presence of TGA1a, oligonucleotides containing TGA1a-binding sites do not significantly affect the stimulated level of transcription. This result indicates that a complex remains committed to the promoter site after initiation and that this complex is used repeatedly during several initiation events. Our study demonstrates for the first time that a plant factor can activate transcription in a human in vitro system and that the activation mechanism of the plant factor is similar to that of mammalian factors.