Abstract Poly(ethylene glycol) hydrogels were synthesized with pendant peptide functionalities to examine the influence of synergistic peptide sequences on osteoblast adhesion, spreading, and function. Specifically, acrylated monomers were prepared that contained the peptide sequence, Arg–Gly Asp (RGD), as well as monomers with RGD plus its synergy site, Pro–His–Ser–Arg–Asn (PHSRN), linked via a polyglycine sequence to recapitulate the native spacing of fibronectin. The colocalized RGD–PHSRN sequence improved osteoblast adhesion, spreading, and focal contact formation when compared to RGD alone. In addition, proliferation, metabolic activity, and levels of alkaline phosphatase production, a common marker for osteoblast function, were statistically higher for the colocalized peptide sequences at 1 day, 1 week, and 2 weeks, when compared to control surfaces. Interestingly, increases were not observed in all areas of cell function, as extracellular matrix (ECM) production was the lowest on gels functionalized with the colocalized peptide sequence. This result was attributed to strong receptor–ligand interactions initiating signal transduction cascades that down-regulate ECM production.