Valvular interstitial cells (VICs) maintain functional heart valve structure and display transient fibroblast and myofibroblast properties. Most cell characterization studies have been performed on plastic dishes; while insightful, these systems are limited. Thus, a matrix metalloproteinase (MMP) degradable poly(ethylene glycol) (PEG) hydrogel system is proposed in this communication as a useful tool for characterizing VIC function in 3D. When encapsulated, VICs attained spread morphology, and proliferated and migrated as shown through real-time cell microscopy. Additionally, fibronectin derived pendant RGD was incorporated into the system to promote integrin binding. As RGD concentration increased from 0 to 2000 microM, VIC process extension and integrin alpha(v)beta(3) binding increased within two days. By day 10, integrin binding was equalized between conditions. VIC morphology and rate of process extension were also increased through decreasing the hydrogel matrix density presented to the cells. VIC differentiation in response to exogenously delivered transforming growth factor-beta1 (TGF-beta1) was also examined within the hydrogel networks. TGF-beta1 increased expression of alpha smooth muscle actin (alphaSMA) and collagen-1 at both the mRNA and protein level by day 2 of culture, indicating myofibroblast differentiation, and was sustained over the course of the study (2 weeks). These studies demonstrate the utility, flexibility, and biological activity of this MMP-degradable system for the characterization of VICs, an important cell population for tissue engineering viable valve replacements and understanding valvular pathobiology.