Our ability to synthesize nanometre-scale chemical species, such as nanoparticles with desired shapes and compositions, offers the exciting prospect of generating new functional materials and devices by combining them in a controlled fashion into larger structures. Self-assembly can achieve this task efficiently, but may be subject to thermodynamic and kinetic limitations: reactants, intermediates and products may collide with each other throughout the assembly time course to produce non-target species instead of target species. An alternative approach to nanoscale assembly uses information-containing molecules such as DNA to control interactions and thereby minimize unwanted cross-talk between different components. In principle, this method should allow the stepwise and programmed construction of target products by linking individually selected nanoscale components-much as an automobile is built on an assembly line. Here we demonstrate that a nanoscale assembly line can be realized by the judicious combination of three known DNA-based modules: a DNA origami tile that provides a framework and track for the assembly process, cassettes containing three independently controlled two-state DNA machines that serve as programmable cargo-donating devices and are attached in series to the tile, and a DNA walker that can move on the track from device to device and collect cargo. As the walker traverses the pathway prescribed by the origami tile track, it sequentially encounters the three DNA devices, each of which can be independently switched between an 'ON' state, allowing its cargo to be transferred to the walker, and an 'OFF' state, in which no transfer occurs. We use three different types of gold nanoparticle species as cargo and show that the experimental system does indeed allow the controlled fabrication of the eight different products that can be obtained with three two-state devices.