The development of methods to transport peptides into cells via a passive mechanism would greatly aid in the development of therapeutic agents. We recently demonstrated that an impermeable fluoresceinated pentapeptide enters the cytoplasm and nucleus of COS 7 cells in the presence of a host-rotaxane by a mechanism that does not depend on an active cell-mediated process. In this report, we further investigate the ability of the host-rotaxane to deliver peptides possessing a wide range of polarities (negatively charged, positively charged, polar, and apolar side chains) into live cells. Only in the presence of the host-rotaxane were the Fl-peptides taken up by COS 7 and ES2 cells. Flow cytometry experiments demonstrated that the level of delivery is largely temperature and adenosine 5'-triphosphate (ATP) independent, and the membranes remain intact. Although the level of transport does depend upon the nature of the side chains, it does not correlate with calculated LogD values, indicating that an additional interaction with the host-rotaxane is modifying the permeability properties of the peptide. The amount of Fl-peptides transported from an aqueous phase into a chloroform phase in the presence of the host-rotaxane correlates with the intensity of cellular fluorescence. Extraction and U-tube studies show that the Fl-peptide can be released from its complex with the host-rotaxane into an aqueous phase, and the host-rotaxane can transport a greater than a stoichiometric amount of an Fl-peptide through a CHCl3 layer. These studies demonstrate the utility of the host-rotaxane in delivering peptides of all polarities across a cell membrane.