Hydrogels are a central component of many approaches to engineer functional tissue replacements. These hydrogels exhibit mechanics similar to native tissue, and cell adhesion and matrix degradation can be readily manipulated. More recently, gene delivery from hydrogels has been investigated as a means to modulate the local microenvironment. We are investigating the design of hydrogels for gene delivery, and this report addresses the role of the adhesive environment on transfection. Previous reports from our laboratory have indicated that fibronectin significantly enhanced gene transfer in the traditional 2D cell culture. To regulate cell adhesion in a hydrogel, we employ a 4-arm PEG backbone, with bifunctional degradable crosslinker, and monofunctional ECM-based adhesion sites. Fibrin hydrogels were employed as a reference hydrogel, as it is a natural material that supports cell adhesion. PEG hydrogels were formed with RGD peptides, fibronectin, or the fibronectin fragment (Fn 7-10). The obtainable ranges of mesh sizes calculated from swelling experiments were between 14 to 27 nm, which would be expected to retain DNA complexes within the network until sufficient degradation allowed for release. Cell migration is supported within the hydrogels, indicating the ability to adhere to the matrix and degrade the hydrogel. Release profiles using radiolabeled DNA indicated that 20% of the encapsulated DNA complexes were released by burst effect, followed by a sustained release for 20 days. Confocal microscopy was used to visual complexes within the hydrogel, and indicated complexes were stable without aggregation. Three configurations of cell and DNA encapsulation were investigated to simulate the multiple uses of hydrogels. The first two approaches involve encapsulating cells within the hydrogel to simulate the use of the hydrogel for in vitro tissue models or a cell transplantation vehicle. These two approaches involve a mixing order of i) cells, DNA, and precursor solution or ii) DNA, precursor solution, and cells, followed by gelation. Finally, DNA and precursor solution were mixed, gelled and cells seeded on top, which simulates cell infiltration into the hydrogel. The first two approaches produced substantially higher cell transgene expression relative to the final approach. ECM has widely been employed to regulate cell function, and these studies indicate that the ECM identity and density can also regulate transgene expression. Our results demonstrate the ability to achieve transfection of cells in vitro while supporting cell proliferation and infiltration, which may be useful for a range of applications.