The interaction of estrogen receptor alpha (ERalpha) with the consensus LXXLL motifs of transcriptional coactivators provides an entry for functional ERalpha inhibition. Here, synthetic cell-permeable LXXLL peptide probes are brought forward that allow evaluation of the interaction of specific recognition motifs with ERalpha in the context of the cell. The probes feature a nona-arginine tag that facilitates cellular entry and induces probe localization in nucleoli. The nucleoli localization provides an explicit tool for evaluating the LXXLL motif interaction with ERalpha. The probes compete with coactivators, bind ERalpha, and recruit it into the nucleoli. The physical inhibition of the ERalpha-coactivator interaction by the probes is shown to be correlated with the inhibition of ERalpha-mediated gene transcription. This chemical biology approach allows evaluating the ERalpha-coactivator interaction and inhibitor binding directly in cells.