Semi-synthetic, proteolytically degradable polymer hydrogels have proven effective as scaffolds to augment bone and skin regeneration in animals. However, high costs due to expensive peptide building blocks pose a significant hurdle towards broad clinical usage of these materials. Here we demonstrate that tri-amino acid peptides bearing lysine (or arginine), flanked by two cysteine residues for crosslinking, are adequate as minimal plasmin-sensitive peptides in poly(ethylene glycol)-based hydrogels formed via Michael-type addition. Substitution of lysine (or arginine) with serine rendered the matrices insensitive to the action of plasmin. This was demonstrated in vitro by performing gel degradation experiments in the presence of plasmin (0.1 U/mL), and in the in vivo situation of regeneration of critical-sized bone defects. When placed as implants into rat calvaria, gels formed from the minimal plasmin substrates showed clear signs of cell infiltration and gel remodeling that coincided with extensive bone formation. (c) 2009 Wiley Periodicals, Inc. J Biomed Mater Res, 2009.