We constructed a single-chain variable fragment miniantibody (G11-scFv) directed toward the transactivation domain of c-Myc, which is fused with the internalization domain Int of Antennapedia at its carboxyl terminus (a cargo-carrier construct). In ELISA experiments, an EC₅₀ for binding saturation was achieved at concentrations of G11-scFv-Int(-) of 10^-8 M. Internalization of a fluoresceinated Fl-G11-scFv-Int(+) construct was observed in intact human cultured cells with confocal microscopy. After 5 h of incubation in medium containing 1 µM Fl-G11-scFv-Int(+) or Fl-G11-scFv-Int(-), fluorescence intensity was determined in individual cells, both for cytoplasmic and nuclear compartments: concentration levels of Fl-G11-scFv-Int(+), relative to the extracellular culture medium concentration, were 4–5 times higher in the cytoplasm, 7–8 times higher in the nucleus, and 10 times higher in the nucleoli. In the same experimental conditions, the Fl-G11-scFv-Int(-) construct was 3–4 times more concentrated outside of the cells than inside. Cell membranes kept their integrity after 5 h of incubation. The antiproliferative activity of our miniantibody was studied on HCT116 cells. Incubation with 4 µM G11-scFv-Int(+) for 4 days induced very significant statistical and biological growth inhibition, whereas Int alone was completely inactive. Miniantibodies capable of penetrating cell membranes dramatically broaden the potential for innovative therapeutic agents and attack of new targets.—Avignolo, C., Bagnasco, L., Biasotti, B., Melchiori, A., Tomati, V., Bauer, I., Salis, A., Chiossone, L., Mingari, M. C., Orecchia, P., Carnemolla, B., Neri, D., Zardi, L., Parodi S. Internalization via Antennapedia protein transduction domain of an scFv antibody toward c-Myc protein.