Abstract Parameters that affect cellular transfection as accomplished by introducing DNA via carriers composed of cationic synthetic amphiphiles, have been investigated, with the aim to obtain insight into the mechanism of DNA translocation. Such insight may be exploited in optimizing carrier properties of synthetic amphiphiles for molecules other than nucleic acids. In the present work, the interaction of vesicles composed of the cationic amphiphile dioleyloxy-propyl-trimethylammonium chloride (DOTMA) with cultured cells was examined. The results show that optimal transfection is dependent on the concentration of lipid, which determines the efficiency of vesicle interaction with the target cell membrane, as well as the toxicity of the amphiphiles towards the cell. A low lipid/DNA ratio prevents the complex from interacting with the cell surface, whereas at a relatively high amphiphile concentration the complex becomes toxic. Translocation efficiency is independent of the initial vesicle size but is affected by the size of the DNA. An incubation time of the DNA/amphiphile complex and cells of approx. 2–4 h is required for obtaining efficient transfection. In conjunction with observations on DNA/amphiphile complex-induced hemolysis of erythrocytes, a mechanism of DNA-entry is proposed which involves translocation of the nucleic acids through pores across the membranes rather than delivery via fusion or endocytosis. Dioleoylphosphatidylethanolamine, a phospholipid frequently used in a mixture with DOTMA (‘lipofectin’) strongly facilitates this pore formation. Translocation of the DNA is effectively prevented when the cells are pretreated with Ca 2+ or pronase. These observations suggest that Ca 2+-sensitive cell surface proteins play a role in amphiphile-mediated DNA translocation.