Antennapedia and other homeoproteins have the unique ability to efficiently translocate across biological membranes, a property that is mediated by the third helix of the homeodomain. To analyze the effects of sequence divergence in the homeodomain, we have compared the cellular uptake efficiencies and interaction properties in a membrane-mimicking environment of four peptides corresponding to the third helix sequence of Antennapedia, Engrailed-2, HoxA-13, and Knotted-1. NMR studies revealed that these peptides adopt helical conformations in SDS micelles. Their localization with respect to the micelle was investigated using Mn²⁺ as a paramagnetic probe. Peptides are positioned parallel to the micelle surface, but subtle differences in the depth of immersion were observed. Using a recently developed method for quantification of CPP cellular uptake based on MALDI−TOF mass spectrometry, all of these peptides were found to translocate into cells but with large differences in their uptake efficiencies. The peptide with the highest uptake efficiency was found to be the least deeply inserted within the micelle, indicating that electrostatic surface interactions may be a major determinant for membrane translocation. A new cell-penetrating peptide derived from Knotted-1 homeodomain with improved uptake properties compared to penetratin is introduced here.