Protein transduction domains (PTDs), such as HIV TAT PTD, have been widely used as delivery tools into living cells. Here we reported for the first time that the helix-loop-helix (HLH) domain of basic helix-loop-helix (bHLH) family was a novel type of PTD. Efficient internalization has been obtained with HLH domains derived from bHLH proteins, NeuroD/BETA2, Neurogenin3, and Mitf, in various cell types including stable cell lines and primary cells. Cellular uptake of HLH PTD was barely or slightly inhibited by the metabolic, phagocytosis, clathrin- or caveolar-endocytosis formation inhibitors, but significantly and substantially reduced by heparin and macropinocytosis inhibitor, which suggested important roles of cell surface glycosaminoglycans and macropinocytosis during the protein transduction. Furthermore, NeuroD proteins still preserved transcription activation after internalized into cells. Our results demonstrated a new motif of PTD different from previous models as cationic residues cluster or amphipathic helix. The HLH domain is also the characteristic of the bHLH family, which implied a large number of PTDs could be discovered in this family to fit different purposes, and some of them could be directly recruited to penetrate cell membrane according to their crucial roles in development such as NeuroD and Ngn3. (c) 2006 Elsevier Inc. All rights reserved.