The third helix of the Antennapedia homeodomain is internalized by cells in culture without requiring a specific receptor. This was demonstrated by showing that a reverse helix and a helix composed of denantiomers still translocate across biological membranes at 4 and 37°C. Additionally, introducing prolines into the structure did not hinder internalization, suggesting that the α-helical structure is not necessary. The data support a translocation process based on direct interactions with membrane phospholipids. The third helix of the homeodomain has been used to deliver biologically active substances to the cytoplasm and nucleus of cultured cells. These findings have physiological implications and open the way to the molecular design of cellular vectors. Homeoproteins are transcription factors involved in important biological processes, particularly during development. The DNA-binding domain of these proteins, the homeodomain, consists of three α-helices. The third helix, called the recognition helix, is involved in DNA binding. The homeodomain of Antennapedia is internalized by cells in culture and is transported to the nucleus where it can interfere with transcription. Internalization occurs at both 4 and 37°C, suggesting it is not receptor-mediated. The region of the homeodomain responsible for internalization has been mapped to its third helix. A 16-amino acid peptide corresponding to the third helix translocates across biological membranes, reaches the cytoplasm, and is conveyed to the nucleus. This peptide has been successfully used as a vector to deliver biologically active compounds to live cells. The mechanism of translocation is not dependent on a specific receptor or the formation of a charged pore. The study suggests that the internalization process may involve the formation of inverted micelles or fluid-phase pinocytosis. The findings have implications for the development of new vector systems for intracellular delivery of pharmacological substances and for understanding the physiological significance of homeodomain internalization.The third helix of the Antennapedia homeodomain is internalized by cells in culture without requiring a specific receptor. This was demonstrated by showing that a reverse helix and a helix composed of denantiomers still translocate across biological membranes at 4 and 37°C. Additionally, introducing prolines into the structure did not hinder internalization, suggesting that the α-helical structure is not necessary. The data support a translocation process based on direct interactions with membrane phospholipids. The third helix of the homeodomain has been used to deliver biologically active substances to the cytoplasm and nucleus of cultured cells. These findings have physiological implications and open the way to the molecular design of cellular vectors. Homeoproteins are transcription factors involved in important biological processes, particularly during development. The DNA-binding domain of these proteins, the homeodomain, consists of three α-helices. The third helix, called the recognition helix, is involved in DNA binding. The homeodomain of Antennapedia is internalized by cells in culture and is transported to the nucleus where it can interfere with transcription. Internalization occurs at both 4 and 37°C, suggesting it is not receptor-mediated. The region of the homeodomain responsible for internalization has been mapped to its third helix. A 16-amino acid peptide corresponding to the third helix translocates across biological membranes, reaches the cytoplasm, and is conveyed to the nucleus. This peptide has been successfully used as a vector to deliver biologically active compounds to live cells. The mechanism of translocation is not dependent on a specific receptor or the formation of a charged pore. The study suggests that the internalization process may involve the formation of inverted micelles or fluid-phase pinocytosis. The findings have implications for the development of new vector systems for intracellular delivery of pharmacological substances and for understanding the physiological significance of homeodomain internalization.