Abstract: Mixed enzyme liquid of cellulase R-10,Pectolyase Y23and mannitol was used to extract the protoplasts from sterilized seedling leaves and endosperm callus of Parthenocissus tricuspidata and P.quenquefolia.The factors affectiong the yield and activities of portoplasts were analyzed and the yield of protoplast from different sources was compared.The protoplasts obtained were cultured by liquid,solid-liquid and solid culture methods.The differentiated cultures were conducted on the callus formed from protoplasts on MS and modified B5 media with different concentrations of NAA,6-BA,2,4-D,PEG,KT,and ZT.The results showed that cellulase R-10 had extremely singificant influences on the extract yield of protoplasts.The suitable enzyme liquid combination for extracting protoplast of P.tricuspidata was:cellulase R-10 0.3%,mannitol 0.6 mol·L-1,with enzymolysis time 8 hours.There existed significant differences among different sources in the yield of protoplast.Only the protoplast from P.quenquefolia endosperm callus could form new callus on soild media.While the protoplasts from other sources had no divistion or disintegrated after forming cell groups containing dozens of cells.The new callus tissues formed from endosperm callus protoplasts of P.quenquefolia were differentiated cultured with over thirty prescriptions,and no differentiation was found after 6-10 subculture.