In this paper, we describe a simple method for fabricating micropatterned polymeric fibrous scaffolds that are capable of controlling the spatial positioning of mammalian cells. Micropatterned scaffolds were prepared by fabricating poly(ethylene glycol) (PEG) hydrogel microstructures on electrospun poly(styrene) (PS) fiber matrices, and produced as free-standing and bidirectionally-porous sheets. Clearly defined hydrogel micropatterns incorporating PS fibers were created using photolithography without any residual hydrogel precursor solution remaining in the PS fiber region. Most of the PS fibers were inserted through the side walls of the hydrogel microstructures, leaving no fiber residue on the top surfaces of hydrogel micropatterns. Since the PEG hydrogel showed non-adhesiveness toward proteins and cells, cells selectively adhered and remained viable within the PS fiber region, thereby creating cellular micropatterns. Finally, possible application of this system to cell-based biosensor was demonstrated using cytotoxicity assay. (C) 2010 Elsevier B.V. All rights reserved.