The repair or replacement of damaged tissues using in vitro strategies has focused on manipulation of the cell environment by modulation of cell‐extracellular matrix interactions, cell‐cell interactions, or soluble stimuli. Many of these environmental influences are easily controlled using macroscopic techniques; however, in co‐culture systems with two or more cell types, cell‐cell interactions have been difficult to manipulate precisely using similar methods. Although microfabrication has been widely utilized for the spatial control of cells in culture, these methods have never been adapted to the simultaneous co‐cultivation of more than one cell type. We have developed a versatile technique for micropatterning of two different cell types based on existing strategies for surface modification with aminosilanes linked to biomolecules and the manipulation of serum content of cell culture media. This co‐culture technique allowed manipulation of the initial cellular microenvironment without variation of cell number. Specifically, we were able to control the level of homotypic interaction in cultures of a single cell type and the degree of heterotypic contact in co‐cultures over a wide range. This methodology has potential applications in tissue engineering, implant biology, and developmental biology, both in the arena of basic science and optimization of function for technological applications. © 1997 John Wiley & Sons, Inc.