An investigation was undertaken into the method of delivery of RGD peptide motifs to adult human mesenchymal stem cells (hMSCs) encapsulated in poly(ethylene glycol) (PEG) hydrogel systems. Previous studies have shown that the viability of hMSCs encapsulated in bio-inert hydrogels, such as PEG-based gels, decreases over time. hMSCs are an adhesive-dependent cell type, requiring attachment sites to maintain their survival and function. The incorporation of tethered RGD peptide motifs was found to sustain a high level of hMSC survival in PEG gels. However, previous reports are largely limited to pendantly tethered RGD in gels; therefore, further investigation into hMSCs' affinity for and response to the contextual presentation of RGD was studied using varying methods of RGD attachment to the PEG gel, as well as delivery of soluble RGD peptides. Studies with encapsulated hMSCs showed that the constrained RGD peptide, bound via two links to the PEG gel, promoted ∼60% cell survival, while tethering the RGD as a pendant group, bound via a single link to the PEG gel, promoted ∼80% cell survival. Interestingly, incorporating a glycine spacer arm to the RGD pendant tether further enhanced survival to ∼88%. Investigations with solubly delivered peptides resulted in a dramatic decrease in cell viability with time, eventually leading to survival that was similar to that of unmodified PEG gels. Integrin staining for αvβ3 and α4, as well as focal adhesion staining, correlates to the trends in hMSC survival for covalently-bound RGD motifs and a loss of viability for the solubly delivered peptide systems. Copyright © 2008 John Wiley & Sons, Ltd.