In the recent molecular and cell biological research, there is an increasing need for labeling of subcellular structures in living cells. Here, we present the use of a fluorescently labeled cell penetrating peptide for fast labeling of nucleoli in living cells of different species and origin. We show that the short peptide with ten amino acids was able to cross cellular membranes and reach the nucleolar target sites, thereby marking this subnuclear structure in living cells. The treatment of cells with actinomycin D and labeling of B23 protein and fibrillarin provided evidence for a localization to the granular component of the nucleolus. The fluorescently conjugated nucleolar marker could be used in combination with different fluorophores like fluorescent proteins or DNA dyes, and nucleolar labeling was also preserved during fixation and staining of the cells. Furthermore, we observed a high stability of the label in long-term studies over 24 h as well as no effect on the cellular viability and proliferation and on rDNA transcription. The transducible nucleolar marker is therefore a valuable molecular tool for cell biology that allows a fast and easy labeling of this structure in living cells.