Cell printing is a promising approach to create organized constructs for tissue engineering applications. We present an automated cell printing microtechnology based on the use of an aqueous two-phase system (ATPS) interfaced with a three-axis motorized system. Cells suspended in the denser aqueous dextran (DEX) phase are loaded into printing tips, which are placed onto the cartridge of the motorized system. Using a computer interface, tips are lowered in the vicinity of a biological surface maintained in the immersion, aqueous polyethylene glycol (PEG) phase to perform a horizontal motion, autonomously dispense their contents onto the surface, and retracted out of the PEG phase. The motorized ATPS technology allows precise spatial and temporal control of the printing process and supports printing fully viable cells. We conduct a systematic study and show that the resolution of ATPS-mediated cellular patterns depends on several factors including the dimensions of the printing tips, lateral speed of tips during horizontal motion, and the loaded volume of the DEX phase in the tips. The finest resolution is ∼300 µm obtained with a tip diameter of 200 µm at a printing tip speed of 16.5 mm/s. Higher speeds result in unstable DEX patterns that break into drops due to capillary instability, and thus are avoided. We also test a number of printing substrates and find that in addition to a cell monolayer, decellularized matrices can serve as a substrate for cell printing with ATPS. Using the principles from the characterization studies, we create duplex prints of cells to demonstrate the potential of this approach for spatio-temporally controlled cell placement. The ATPS printing microtechnology will be a step forward toward developing well-organized, three-dimensional tissue constructs. Biotechnol. Bioeng. 2014;111: 404–412. © 2013 Wiley Periodicals, Inc.