The yeast trans-activator protein GAL4, when expressed in HeLa cells, stimulates transcription from several class B (II) eukaryotic promoters containing GAL4 binding sites either as the full UASG or as synthetic 17-mers. The characteristics of this activation are indistinguishable from those of the SV40 enhancer. Transcription was similarly stimulated from either complex promoter regions containing multiple upstream elements or from a simple promoter region composed of only a TATA box. Addition of a 17-mer GAL4 binding site to the SV40 enhancer resulted in a synergistic enhancement of transcription in the presence of GAL4. Furthermore, chimeras of the human estrogen receptor DNA binding domain and either GAL4 or GCN4 activating "acidic" regions can activate a promoter region controlled by an estrogen-responsive enhancer. Together, these data indicate that the molecular mechanisms responsible for transcriptional enhancement have been conserved from yeast to man.