Loligomers are multitasking, peptide-based shuttles that are able to penetrate cells and self-localize into distinct cellular compartments. In particular, loligomer 4 incorporates internalization and nuclear import sequences as well as reporter groups. The intracellular routing of loligomer 4 was analyzed by microscopy and flow cytometry, to define and demonstrate localization events. Electron micrographs of CHO cells exposed to a biotinylated derivative of loligomer 4 as well as confocal images of CHO cells treated with rhodamine-labeled loligomer 4 indicate their presence in the cytosol, endocytic vesicles, and the nucleus of CHO cells. Loligomer 4 accumulates irreversibly inside cells. Uptake of loligomer 4 by six mammalian cell lines (Daudi, EL4, CHO, COS-7, VERO, and HeLa) was proven by flow cytometry, establishing the generality of the principle. Cells presented as monolayers typically were less able to endocytose the construct than cells grown in suspension. Cellular accumulation of loligomer 4 varied between cell lines with COS-7 and VERO cells showing the highest level of uptake. Plasmids harboring reporter genes could be transported efficiently inside CHO cells, suggesting that loligomer 4 either alone or noncovalently associated with large macromolecules can effectively reach the nucleus of cells. In summary, loligomer 4 constructs provide a simple synthetic platform for the design of guided intracellular agents.