Phosphopeptide-cellular uptake has been studied with a unique combination of tools designed to quantitate this phenomena and to understand properties that contribute to transmembrane penetration. High-affinity src-homology domain (SH2) hexapeptides for the phosphatidyl inositol 3-kinase system were used to judge cell penetration using red blood cells--a model system for the study of transmembrane cellular uptake. Hexapeptides without phosphate groups and devoid of charged residues poorly entered cells. N-terminal modification with bulky hydrophobic groups enhanced partitioning into octanol, an index of hydrophobicity, and allowed certain non-phosphorylated peptides to pass into red cells. However, tyrosine phosphorylation of hexapeptides markedly decreased octanol-water partitioning and completely eliminated cellular uptake. Inclusion of ion-pairing agents that masked the phosphate hydrophilic character enabled partitioning of phosphopeptides into octanol and achieved cellular uptake. This effect was demonstrated using fluorescent derivatives of phosphopeptides and CV1 cells in culture. The results validate the concept of facilitating cell entry by charge masking and open the way to future refinements of this principle. Various penetration techniques are compared and discussed in the context of maximizing cellular viability.