High costs of commercially available lytic enzymes oblige to searching for new methods of isolation of protoplasts from filamentous fungi and yeast cells. The aim of work was the effect of the culture filtrate from Trichoderma viride F-19 on the protoplasts formation of Trichoderma reesei M-7 in comparison with commercially available preparations of cell-wall lytic enzymes. The yields of protoplasts obtained with culture filtrates of Trichoderma viride F-19 and Lytic Enzymes from Cytophage were comparable. Less number of protoplasts has been estimated after incubation of cells with Lyticase. In spite of higher yield of protoplasting, protoplasts derived from culture filtrates of Trichoderma viride F-19 and Lytic Enzymes from Cytophage gave a worse regeneration frequency then Lyticase – derived protoplasts.