Poly(vinyl alcohol) (PVA) hydrogels interacting with DNA mediated by hydrogen bonds (PVA/DNA hydrogel) were developed using ultra-high pressure (UHP) technology. The goal was to create a new method of gene delivery by controlled release of DNA. Mixed solutions of DNA and PVA at various concentrations were pressurized at 10 000 atmospheres at 37°C for 10 min. PVA/DNA hydrogels with good formability were produced at PVA concentrations of more than 5% w/v. The presence of DNA in the obtained hydrogels was confirmed by spectroscopic analysis and nucleic acid dye staining. DNA release from the hydrogels was investigated using PVA/DNA hydrogel samples of 5% and 10% w/v formed by UHP treatment or by conventional freeze-thaw methods. The DNA release curves from both types of samples showed a rapid phase in the initial 15 h followed by a sustained release phase. However, there was a difference in the amount of DNA released. Less DNA was released by the pressurized hydrogels than by the freeze-thaw hydrogels. Also, the cumulative amount of DNA released decreased as the PVA content in the hydrogels increased. These results indicate that DNA release from the hydrogels can be modulated by changing the preparation method and the PVA content. Furthermore, it was demonstrated that DNA release could be controlled by varying the amount and duration of pressurizing used to form the hydrogels. Intact fractions of plasmid DNA released from the hydrogels were separated by agarose gel electrophoretic analysis. These results suggest that, using controlled release, DNA from PVA/DNA hydrogels formed by UHP treatment can be transfected into cells. © 2007 The Japanese Society for Artificial Organs.