Methods for culturing mammalian cells ex vivo are increasingly needed to study cell and tissue physiology and to grow replacement tissue for regenerative medicine. Two-dimensional culture has been the paradigm for typical in vitro cell culture; however, it has been demonstrated that cells behave more natively when cultured in three-dimensional environments. Permissive, synthetic hydrogels and promoting, natural hydrogels have become popular as three-dimensional cell culture platforms; yet, both of these systems possess limitations. In this perspective, we discuss the use of both synthetic and natural hydrogels as scaffolds for three-dimensional cell culture as well as synthetic hydrogels that incorporate sophisticated biochemical and mechanical cues as mimics of the native extracellular matrix. Ultimately, advances in synthetic–biologic hydrogel hybrids are needed to provide robust platforms for investigating cell physiology and fabricating tissue outside of the organism. Biotechnol. Bioeng. 2009;103: 655–663. © 2009 Wiley Periodicals, Inc.