Our goal is to define the properties of cell-impermeable cargo molecules that can be delivered into cells by pH (Low) Insertion Peptide (pHLIP), which can selectively target tumors in vivo based on their acidity. Using biophysical methods and fluorescence microscopy, we show that pHLIP can successfully deliver polar and membrane-impermeable cyclic peptides linked to its C terminus through the membranes of lipid vesicles and cancer cells. Our results also indicate that the translocation of these cargo molecules is pH dependent and mediated by transmembrane helix formation. Since a broad range of cell-impermeable molecules is excluded from discovery efforts because they cannot traverse membranes on their own, we believe that pHLIP has the potential to expand therapeutic options for acidic tissues such as tumors and sites of inflammation.