Introduction: Duramycin through binding with phosphatidylethanolamine (PE) has shown potential to be an effective anti-tumour agent. However its mode of action in relation to tumour cells is not fully understood. Methods: PE expression on the surface of a panel of cancer cell lines was analysed using duramycin and subsequent antibody labelling then analysed by flow cytometry. Cell viability was also assessed via flow cytometry using annexin V and propidium iodide (PI). Calcium ion (Ca²⁺) release by tumour cells in response to duramycin was determined by spectrofluorometry following incubation with Fluo-3, AM. Confocal microscopy was performed on the cancer cell line AsPC-1 to assess real time cell response to duramycin treatment. Results: Duramycin was able to detect cell surface PE expression on all 15 cancer cell lines screened, which was shown to be duramycin concentration dependent. However higher concentrations induced necrotic cell death. Duramycin induced calcium ion (Ca²⁺) release from the cancer cell lines also in a concentration and time dependent manner. Confocal microscopy showed an influx of PI into the cells over time and induced morphological changes. Conclusion: Duramycin induces Ca²⁺ release from cancer cell lines in a time and concentration dependent relationship