In this study, in vitro and in vivo transfection of temperature-sensitive, polycationic poly(N-isopropylacrylamide) and polyethyleneimine copolymers (poly(NIPA)/PE125L) were performed. Copolymer and copolymer-plasmid DNA (pDNA) complexes were positively charged as +7.6 and +12.8, respectively. Gel retardation assay confirmed good complex formation and release of plasmid DNA in response to temperature and pH. Cytotoxicity tests showed at least 80% smooth muscle cell (SMC) viability. The uptake of the complexes by SMCs was quite high; however, the best gene expression efficiency achieved with the copolymeric vectors was about 30% with the complex prepared with a polymer: plasmid ratio of 6. Gene expression efficiency was enhanced up to 50% by changing the temperature from 37 degrees C to 28 degrees C. Preliminary in vivo studies were performed above and below lower critical solution temperature (LCST) in lung, heart, liver, kidney, muscle and also subcutaneously in 5 week-old mice. The gene expression ratio was higher in lung, tibial muscle and subcutaneously than in other tissues (heart, liver and kidney) above LCST. Then, temperature decrease caused an increase in the amount of gene expression in tibial muscle and subcutaneously, revealing the contribution of temperature-sensitivity on DNA release and gene expression. Copyright (c) 2007 John Wiley & Sons, Ltd.