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Microfabrication tools have been extensively utilized to define and quantify the role of the cellular microenvironment on cell fate and function on two-dimensional surfaces. Exploring structure/function relationships in three-dimensional (3D) tissues will similarly require tools to control cellular organization in 3D. Our aim was to develop a versatile 3D patterning tool compatible with many biomaterials and cell types. Here, we present the development and characterization of a model system that utilizes dielectrophoretic (DEP) forces to rapidly organize a variety of cells within solid hydrogel slabs. Our data indicate that embedded cells remain largely viable and maintain differentiated function over long periods of time. DEP patterning is also compatible with next-generation biomaterials, and may become a useful and versatile tool for investigating structure/function relationships in tissue engineering.