New poly(vinyl alcohol) (PVA) derivatives containing different pendant chemoselective functionalities have been prepared for the in situ formation of hydrogels at physiological conditions. Particularly, incorporations of thiol, cysteine 1,2-aminothiol, and aminooxy side chains to PVA were performed for the first time by direct coupling of the protected nucleophilic functionalities to PVA's hydroxyl groups via carbamate linkages followed by acidic deprotection. In the second approach, PVA was first derivatized to a low degree (3%) with amino groups which were used to quantitatively react with different N-hydroxysuccinimide esters of carboxylic acids containing maleimide, a-iodoacetyl, or acrylate thiophilic functionalities. The utility of the amino-derivatized PVA was also demonstrated for further functionalization with semicarbazide terminated side chains. The ability of the new PVA derivatives to act as multifunctional cross-linking agents was examined in the course of in situ cross-linking reactions with hyaluronic acid carrying aldehyde groups. Use of multi functional PVA cross-linkers was shown to give short gelation times, i.e., within half a minute, which is critical for clinical applications. The hyaluronan hydrogels were enzymatically degradable as evidenced by the results of in vitro degradation by hyaluronidase. Moreover, these hydrogels were found to be nontoxic to human dermal fibroblasts. Hence, PVA-based multifunctional cross-linkers can extend the scope of in situ preparation and properties of hydrogel-based synthetic mimics of extracellular matrix as compared with well established bifunctional poly(ethylene glycol) analogs.