Many eukaryotic transcriptional activator proteins contain a DNA-binding domain that interacts with specific promoter sequences and an acidic activation region that is required to stimulate transcription. Transcriptional enhancement by such activator proteins is often synergistic and promiscuous; promoters containing multiple binding sites for an individual protein or even for unrelated proteins can be 10-100 times more active than promoters with single sites. It has been suggested that such synergy reflects a nonlinear response of the basic transcription machinery to the number and/or quality of acidic activation regions. Here, we determine the transcriptional activity of Jun-Fos heterodimers containing one or two GCN4 acidic activation regions on promoters containing one or two Ap-1 target sites. Surprisingly, heterodimers with one or two acidic regions activate transcription with similar efficiency and are equally synergistic (10- to 15-fold) on promoters containing two target sites. Thus, transcriptional synergy does not depend on the number of acidic activation regions but rather on the number of proteins bound to the promoter. This suggests that synergy is mediated either by cooperative DNA binding or by alternative mechanisms in which the DNA-binding domain plays a more direct role in transcription (e.g., changes in DNA structure, nucleosome displacement, or direct interactions with the transcriptional machinery).