alpha-N-Acetyl-galactosaminidase was purified from several commercially available Aspergillus niger enzyme fractions. By using GalNAc-alpha-pNP as the substrate, a K-m = 0.23 mM and V-max = 210 mu mol/min mg protein and a pH-optimum in the range of 2.6-3.2 were determined. These enzyme preparations were tested under conditions of reverse hydrolysis with unprotected L-serine as the acceptor. Incubation conditions in the preparative synthesis of GalNAc-alpha-(1 --> O)-serine were optimized with respect to incubation time and pH. By using 80 U of enzyme/mmol of starting material at pH 3.5, thermodynamic equilibration of the incubation mixture was reached after 4 days. To scale up the synthesis to the gram-scale, a new purification procedure was developed based on column chromatography on activated carbon. Because the starting material N-acetyl-galactosamine is itself an expensive component, we also examined the recovery of unconverted galactosamine. Recovery rates were determined to be in the range of 60-70%.