The link between a growing number of human diseases and misregulation of gene expression has spurred intense interest in artificial transcriptional activators that could be used to restore controlled expression of affected genes. To expand the repertoire of activation domains available for the construction of artificial transcriptional regulators, a selection strategy was used to identify two unique activation domain motifs. These activation domains bear little sequence homology to endogenous counterparts and bind to unique sites within the transcriptional machinery. A comparison with two well-characterized activation domains, VP2 and P201, demonstrated for the first time that functional potency is not solely dictated by binding affinity. Finally, the selection strategy described is readily applicable to the identification of small molecule activation domains.