Submicron lipid vesicles (nanoliposomes) are being used as carriers of bioactive compounds. In addition, complexes of nanoliposomes and nucleic acids (nanolipoplexes) are promising tools for the treatment of cancer, and viral and genetic disorders. Toxicity of some of these formulations, however, still remains a concern in their clinical utilisation. To address this problem, anionic liposomes were prepared by two different techniques, the conventional thin-film method, and the heating method (HM), in which no volatile organic solvent or detergent is used. An anionic nanolipoplex was constructed by incorporating plasmid DNA (pcDNA3.1/His B/lacZ) into the HM-nanoliposomes by the mediation of calcium. The toxicity of the nanoliposomes, with and without plasmid and Ca2+, was assessed using a human bronchial epithelial cell line (16HBE14o-) in the presence of serum. Cytotoxicity evaluations performed by two different assays (i.e. NRU and MTT) indicated that HM-nanoliposomes were completely non-toxic in the cell-line tested, whereas conventional liposomes revealed significant levels of toxicity. This may be due to the presence of trace amounts of chloroform and/or methanol applied during their preparation. Similar results were obtained for ..