Protein PEGylation has been successfully applied in pharmaceuticals and more recently in biomaterials development for making bioactive and structurally versatile hydrogels. Despite many advantages in this regard, PEGylation of proteins is also known to alter biological activity and modify biophysical characteristics in ways that may be detrimental to cells. The aim of this study was to evaluate the relative loss of biological compatibility associated with PEGylating a fibrinogen precursor into a hydrogel scaffold, in comparison to thrombin cross-linked fibrin hydrogels. Specifically, we investigated the consequences of conjugating fibrinogen with linear polyethtylene glycol (PEG) polymer chains (10 kDa) on the ability to cultivate neonatal human foreskin fibroblasts (HFFs) in 3-D. For this purpose, thrombin cross-linked fibrin (TCL-Fib) and PEGylated fibrinogen (PEG-Fib) gels were prepared with HFFs and cultured for up to seven days. The benchmark biological compatibility test was based on a combined assessment of cellular morphology, proliferation, actin expression, and matrix metalloproteinase (MMP) expression in the 3-D culture systems. The results showed correlations between modulus and proteolytic biodegradation in both materials, but no correlation between the mechanical properties and the ability of HFFs to remodel the microenvironment. A slight reduction of actin, MMPs, and spindled morphology of the cells in the PEG-Fib hydrogels indicated that the PEGylation process altered the biological compatibility of the fibrin. Nevertheless, the overall benchmark performance of the two materials demonstrated that PEGylated fibrinogen hydrogels still retains much to the inherent biofunctionality of the fibrin precursor when used as a scaffold for 3-D cell cultivation. Copyright © 2011 Elsevier Ltd. All rights reserved.