In this paper, the gypenoside-alpha-(1-->6)-L-rhamnosidase from microorganisms was purified and characterized. The enzyme hydrolyzed the 20-C, alpha-(1-->6)-L-rhamnoside of gypenoside-5 to produce ginsenoside Rd, but did not hydrolyzed the alpha-rhamnoside of ginsenoside Rg2, and only partially hydrolyzed the alpha-rhamnoside of p-nitrophenyl-alpha-L-rhamnoside (pNPR) which was a hemi-cellulase substrate. The enzyme was purified to homogeneity by SDS polyacrylamide gel electrophoresis, and its molecular weight was about 68 kDa. The optimum temperature of enzyme reaction was 50degreesC, and the optimum pH was 5. Metal ions activated the gypenoside-alpha-(1-->6)-L-rhamnosidase activity. (C) 2003 Elsevier Ltd. All rights reserved.