In order to improve the quantity and viability of protoplasts released from the mycelia of Aspergillus oryzae HN3042 and Aspergillus niger CICC2377,the effects of carbon resources,mycelia age,osmotic stabilizers,enzyme concentration,incubation time and regeneration media on the release and regeneration of protoplasts were investigated,and the parameters of asymmetric inactivation for the protoplasts were analyzed.Afterwards,suitable conditions for protoplast release were determined.The results indicate that,with MPY consisting of maltose,polytephone and yeast extract as the submerged medium,0.7mol/L NaCl combined with 0.4mol/L sorbitol as the osmo-tic stablizer,and with hypertonic soybean-extract solid medium as the regeneration medium,the released protoplasts show a regeneration efficiency of above 30%.Moreover,according to the asymmetric inactivation curves,it is found that both Aspergillus oryzae HN3042 and Aspergillus niger CICC2377 protoplasts are in critical inactivation stage when they are respectively under the irradiation of 15W ultraviolet from 13cm vertically away for 15min or in a 65℃ water bath for 10min.