The authors have used electrospun polyethylene oxide nanofibers as sacrificial templates to form nanofluidic channels in polydimethylsiloxane (PDMS). By depositing fibers on silicon templates incorporating larger structures, the authors demonstrate that these nanochannels can be integrated easily with microfluidics. They use fluorescence microscopy to image channels filled with dye solution. The utility of the hybrid micro- and nanofluidic PDMS structures for single molecule observation and manipulation was demonstrated by introducing single molecules of lambda-DNA into the channels. This nanofabrication technique allows the simple construction of integrated micro- and nanofluidic PDMS structures without lithographic nanofabrication techniques. (C) 2008 American Vacuum Society.