The 60-amino acid long homeodomain of Antennapedia crosses biological membranes by an energy-independent mechanism, a phenomenon abolished by directed mutagenesis within the polypeptide C-terminal region. This finding led us to study the internalization of several chemically synthesized peptides derived from the third helix of the homeodomain. We report here that a polypeptide of 16 amino acids in length corresponding to the third helix of the homeodomain deleted of its N-terminal glutamate is still capable of translocating through the membrane. A longer peptide of 20 amino acids also translocates, whereas shorter peptides (15 amino acids) are not internalized by the cells. As is also the case for the entire homeodomain, the 20- and 16-amino acid long peptides are internalized at 4 degrees C, suggesting an energy-independent mechanism of translocation not involving classical endocytosis. The two translocated peptides can be recovered, intact, within the cells, strongly suggesting that they are not targeted to the lysosomal compartment. Finally, substitution of two tryptophans by two phenylalanines strongly diminishes translocation, raising the possibility that the internalization of the third helix is not solely based on its general hydrophobicity.