Artificial nanotransport systems inspired by intracellular transport processes have been investigated for over a decade using the motor protein kinesin and microtubules. However, Only unidirectional cargo transport has been achieved for the purpose of nanotransport in a microfluidic system. Here, we demonstrate bidirectional nanotransport by integrating kinesin and dynein motor proteins. Our molecular system allows microtubule orientation of either polarity in a microfluidic channel to construct a transport track. Each motor protein acts as a nanoactuators that transports microspheres in opposite directions determined by the polarity of the oriented microtubules: kinesin-coated microspheres move toward the plus end of microtubules, whereas dynein-coated microspheres move toward the minus end. We demonstrate both unidirectional and bidirectional transport using kinesin- and dyneincoated microspheres on microtubules oriented and glutaraldehyde-immobilized in a microfluidic channel. Tracking and statistical analysis of microsphere movement demonstrate that 87-98% of microspheres move in the designated direction at a mean velocity of 0.22-0.28 mu m/s for kinesincoated microspheres and 0.34-0.39 mu m/s for dynein-coated microspheres. This bidirectional nanotransport goes beyond conventional unidirectional transport to achieve more complex artificial nanotransport in vitro. Biotechnol. Bioeng. 2008;101: 1-8. (C) 2008 Wiley Periodicals, Inc.