Templates constructed from the wheat Em and maize rab28 promoters are efficiently and accurately transcribed in the well-characterized cell-free transcription system prepared from HeLa nuclei. Deletion analysis of the Em promoter indicates that a G-box (CACGTG) element (Em1b) is required for transcription. USF, a Myc transcription factor in HeLa nuclear extracts, activates transcription by binding to Em1b, as shown by the ability of an antibody raised against USF to inhibit transcription and to interfere with Em1b complex formation in an electrophoretic mobility shift assay. The addition of the recombinant Viviparous1 protein from maize to HeLa nuclear extracts specifically stimulated transcription of the Em promoter but was dependent on the presence of USF in the extract. In USF-depleted extracts, the addition of recombinant EmBP1, a basic leucine zipper transcription factor from wheat, activated transcription through Em1b as well as from a similar G-box in the adenovirus major late promoter. Our study demonstrates that the basic transcriptional apparatus in HeLa nuclear extract supports transcription from plant promoters and can be used to assay the function of certain plant nuclear proteins, thereby helping to determine their effects on transcription.