Poly(vinyl acetate), PVAc, is an industrially important polymer used in adhesives, textile fibers, paper products and coating materials. This paper reports catalytic activities of cutinases from Humicola insolens (HiC), Pseudomonas mendocina (PmC), and Fusarium solani for PVAc hydrolysis. PVAc was distributed as thin films throughout highly porous styrene divinyl benzene beads with known surface area. Cutinase activity for PVAc deacetylation was assayed using a pH-stat to measure NaOH consumption versus time and PVAc concentration was expressed as square meter per milliliter. HiC had maximum initial activity at 70 degrees C whereas PmC and FsC performed best at 50 and 40 degrees C, respectively. To limit background chemical hydrolysis, kinetic studies for all three cutinases were performed at pH 7.5. Initial activities for each cutinase at their optimal reaction temperatures were fit by the Michaelis-Menten kinetic model. PmC had the highest affinity for PVAc with K-m of 20 in m(2).mL(-1). Furthermore, HiC and FsC had the highest and lowest catalytic efficiency values, 102 and 20 L.h(-1).m(-2), respectively. Study of cutinase stability showed that, within 96 h at 40 degrees C, FsC lost 93% of its initial activity. In contrast, incubations of HiC and PmC retained 67 and 47% of their activities during incubations for 192 h at 70 and 50 degrees C, respectively. For HiC, % yields of water-soluble copolymer after incubations for 144 and 192 h are 13 and 14%, respectively. The mol % of VAc in P(VAc-co-VOH), solubilized during incubations for 24, 144, and 192 h, is 30, 19, and 6%, respectively. Thus, HiC can deacetylate PVAc to high extents. At 24 h, where P(VAc-co-70 mol% VOH) was isolated from the aqueous phase, the sequence distribution along chains determined by H-1 NMR was found to be highly blocky (eta = 0.36 +/-0.06).