We find that pH-(low)-insertion-peptide (pHLIP)-facilitated translocation of phalloidin, a cell-impermeable polar toxin, inhibits the proliferation of cancer cells in a pH-dependent fashion. The monomeric pHLIP inserts its C terminus across a membrane under slightly acidic conditions (pH 6-6.5), forming a transmembrane helix. The delivery construct carries phalloidin linked to its inserting C terminus via a disulfide bond that is cleaved inside cells, releasing the toxin. To facilitate delivery of the polar agent, a lipophilic rhodamine moiety is also attached to the inserting end of pHLIP. After a 3 h incubation at pH 6.1-6.2 with 2-4 μM concentrations of the construct, proliferation in cultures of HeLa, JC, and M4A4 cancer cells is severely disrupted (> 90% inhibition of cell growth). Treated cells also show signs of cytoskeletal immobilization and multinucleation, consistent with the expected binding of phalloidin to F actin, stabilizing the filaments against depolymerization. The antiproliferative effect was not observed without the hydrophobic facilitator (rhodamine). The biologically active delivery construct inserts into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipid bilayers with an apparent pK(a) of ∼6.15, similar to that of the parent pHLIP peptide. Sedimentation velocity experiments show that the delivery construct is predominantly monomeric (> 90%) in solution under the conditions employed to treat cells (pH 6.2, 4 μM). These results provide a lead for antitumor agents that would selectively destroy cells in acidic tumors. Such a targeted approach may reduce both the doses needed for cancer chemotherapy and the side effects in tissues with a normal pH.