Synthetic molecules capable of activating the expression of specific genes are of great interest as tools for biological research and, potentially, as a novel class of pharmaceutical agents. It has been demonstrated previously that such synthetic transcription factor mimics (STFMs) can be constructed by connecting a sequence-specific DNA-binding module to a molecule capable of binding to the transcriptional machinery via a suitable linker. These chimeras mimic the two basic properties of native transcription factors, which are able to recognize a promoter sequence specifically and to recruit the transcriptional machinery to that promoter. However, none of the compounds of this type reported to date have been shown to function in living cells. We report here the first example of a cell-permeable STFM that activates the transcription of a reporter gene in mammalian cells. The compound is composed of a cell-permeable coactivator-binding peptoid fused to a DNA-binding hairpin polyamide. The peptoid was identified by screening a combinatorial library of approximately 50,000 compounds for binding to the KIX domain of the CREB-binding protein (CBP), a mammalian transcription coactivator. When incubated with cultured HeLa cells carrying a luciferase reporter plasmid bearing several hairpin polyamide-binding sites, a 5-fold increase in luciferase expression was observed. These experiments set the stage for the identification of hairpin polyamide-peptoid conjugates that are targeted to native genes.