Misregulated transcription is linked to many human diseases, and thus artificial transcriptional activators are highly desirable as mechanistic tools and as replacements for their malfunctioning natural counterparts. We previously reported two artificial transcriptional activation domains obtained from synthetic peptide libraries screened for binding to the yeast transcription protein Med15(Gal11). Here we demonstrate that the transcriptional potency of the Med15 ligands is increased through straightforward structural alterations. These artificial activation domains upregulate transcription via specific Med15 binding interactions and do not function in mammalian cells, which lack Med15. This functional specificity stands in contrast to most natural or artificial activation domains that function across all eukaryotic cell types. The results indicate that the screening strategy holds excellent promise for identifying peptide and small molecule transcriptional activators that function by unique mechanisms with advantageous specificity properties.