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Kinesins are molecular motors that transport intracellular cargo along microtubules and provide a model system for force generation that can be exploited for biomotor powered nano- and micro-machines. To use this biological system for microscale transport, the most common approach is to reverse the biological geometry and move microtubules along surfaces functionalized with kinesin motors. The microtubules then become potential transport vehicles for sensors and lab-on-a-chip applications. A key requirement for extracting useful work from this system is confinement and control of microtubule movements over kinesin-coated surfaces. The open channel approaches used to date are limited because microtubules that lose contact with the kinesin motors rapidly diffuse away. As a step toward making stand-alone devices incorporating kinesin motors and microtubules, we have developed methods to fabricate capped channels that provide three-dimensional microtubule confinement. We first tested the activity of kinesin motors on a range of surfaces and found that motors were functional on a number of hydrophilic surfaces and nonfunctional on hydrophobic surfaces. In this work, SU-8 photoresist is used to fabricate open channels and a layer of bisbenzocyclobutene (BCB) or dry-film photoresist is used to encapsulate the channels. To allow sample introduction, we fabricate a hierarchical series of microfluidic channels. In this approach, macroscale (∼250-μm) channels in glass or silicon substrates are used to hold fine-gauge stainless steel tubing and allow connection to various fluid sources and intermediate scale (∼50-μm) channels fabricated in thick (∼50-μm) dry-film photoresist are used to connect the macroscale channels to microscale (1-15-μm) SU-8 photoresist channels. This paper is the first demonstration of kinesin-based microtubule transport in enclosed microfluidic channels and provides an important step toward packaging these biomolecular motors into functional devices.