We have previously evaluated and reported numerous classes of linear and cyclic peptides containing hydrophobic and hydrophilic segments for intracellular delivery of multiple molecular cargos. Herein, a combination of histidine and tryptophan amino acids were designed and evaluated for their efficiency in intracellular delivery of cell-impermeable phosphopeptides and the anti-HIV drug, emtricitabine. Two new decapeptides, with linear and cyclic natures, both containing alternate tryptophan and histidine residues, were synthesized using Fmoc/tBu solid-phase chemistry. The peptides were characterized and purified by using matrix-assisted laser desorption/ionization (MALDI) spectroscopy and high-performance liquid chromatography (HPLC), respectively. These peptides did not show significant toxicity up to 100 µM in ovarian cancer (SK-OV-3) and leukemia cancer (CCRF-CEM) cells. Furthermore, the cellular uptake of a fluorescence (F’)-labeled cell-impermeable phosphopeptide (F’-GpYEEI) was enhanced in the presence of linear (WH)₅ and cyclic [WH]₅ by 2- and 8-fold, respectively, compared to the uptake of the phosphopeptide alone. The cellular uptake was not significantly changed in the presence of endocytosis inhibitors. Furthermore, the intracellular uptake of the fluorescently-labeled anti-HIV drug, emtricitabine (F’-FTC), by linear (WH)₅ and cyclic [WH]₅ in SK-OV-3 cancer cell lines was found to be enhanced by 3.5- and 9-fold, respectively, compared to that of the drug alone. Fluorescent uptake experiments confirmed the localization of F’-GpYEEI-loaded cyclic [WH]₅ intracellularly in the SK-OV-3 cancer cell line after 3 h of incubation. Thus, these data demonstrated that [WH]₅ containing tryptophan and histidine enhanced the cellular uptake of F’-GpYEEI and emtricitabine.