Pectolyase Y23, a new maceration enzyme from Aspergillus japonicus, combined with Cellulase Onozuka R10 liberated protoplasts from tobacco mesophyll in less than 25 min. The protoplasts divided to form cell colonies. This combination of enzymes also liberated protoplasts from mesophyll of rice and oat. The enhanced macerating activity of Pectolyase Y23 was found to be due not only to the high activity of pectin lyase and polygalacturonase, but also to a factor which stimulated maceration.