Cell-penetrating peptides present an attractive and efficient tool for the delivery of a variety of cell impermeable cargoes across the cellular membrane. Cell-penetrating peptides usually consist of short basic peptide sequences that are internalized by a variety of cell lines. Most cell-penetrating peptides lack cell specificity, however, which greatly limits their use as efficient therapeutic agents. Herein, we present two cell-penetrating peptides displaying a type II polyproline helical backbone that are functionalized to contain six cationic moieties and two distinctive hydrophobic functionalities, namely isobutyl or benzyl groups. The uptake efficiency of these cationic amphiphilic polyproline helices was studied in seven different cell lines, six cancerous (MCF-7, HOS, HT1080, HeLa, KB-FD, KB3-1) and one non-cancerous (WI 38). The cationic amphiphilic polyproline helix P11LRR at 50 microM showed high specificity toward MCF-7 breast cancer cells. Co-culture experiments with P11LRR demonstrated almost exclusive internalization by MCF-7 cells and not WI38. The replacement of the isobutyl hydrophobic group with a benzyl moiety resulted in a shift in uptake efficiency and specificity across some cell lines. These results demonstrate that the type of hydrophobic residues utilized in the creation of cell-penetrating peptides can strongly influence the extent and specificity of cellular internalization.