The patterned deposition of cells and biomolecules on surfaces is a potentially useful tool for in vitro diagnostics, high‐throughput screening, and tissue engineering. Here, we describe an inexpensive and potentially widely applicable micropatterning technique that uses reversible sealing of microfabricated parylene‐C stencils on surfaces to enable surface patterning. Using these stencils it is possible to generate micropatterns and copatterns of proteins and cells, including NIH‐3T3 fibroblasts, hepatocytes and embryonic stem cells. After patterning, the stencils can be removed from the surface, plasma treated to remove adsorbed proteins, and reused. A variety of hydrophobic surfaces including PDMS, polystyrene and acrylated glass were patterned using this approach. Furthermore, we demonstrated the reusability and mechanical integrity of the parylene membrane for at least 10 consecutive patterning processes. These parylene‐C stencils are potentially scalable commercially and easily accessible for many biological and biomedical applications. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res 2008