Understanding cell behavior inside complex, three‐dimensional (3D) microenvironments with controlled spatiotemporal patterning of physical and biochemical factors would provide significant insights into the basic biology of organ development and tissue functions. One of the fundamental limitations in studying such behavior has been the inability to create patterned microenvironments within 3D scaffold structures. Here a simple, layer‐by‐layer stereolithography (SL) method that can precisely pattern ligands, extracellular‐matrix (ECM) components, and growth factors, as well as controlled release particles inside a single scaffold, has been developed. The process also allows fabrication of predesigned internal architectures and porosities. Photocrosslinkable poly(ethylene glycol) dimethacrylate (PEGDMA) was used as the basic structural component of these microfabricated scaffolds. PEG acrylates, covalently modified with the cell adhesive peptide arginine‐glycine‐aspartic acid (RGD) or the ECM component heparan sulfate, was incorporated within the scaffolds to facilitate cell attachment and to allow spatial sequestration of heparan‐binding growth factors. Fluorescently labeled polymer microparticles and basic fibroblast growth factor (FGF‐2) were chosen to illustrate the capability of SL to spatiotemporally pattern scaffolds. The results demonstrate that a precise, predesigned distribution of single or multiple factors within a single 3D structure can be created, and specific internal architectures can be fabricated. Functionalization of these scaffolds with RGD is demonstrated, and heparan sulfate allows efficient cell attachment and spatial localization of growth factors. Such patterned scaffolds might provide effective systems to study cell behavior in complex microenvironments and could eventually lead to engineering of complex, hybrid tissue structures through predesigned, multilineage differentiation of a single stem‐cell population. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2005