SUMMARY Spherical, osmotically sensitive protoplasts were liberated from the mycelium of Schizophyllum commune through the action of an extracellular enzyme prepara- tion from the culture filtrate of Trithoderma viride grown on hyphal walls of the former organism. The conditions for obtaining stable protoplasts were determined. Maximum numbers of protoplasts were released from young growing mycelium by using MgS04 or KCl at an osmotic potential between - 12-8 and - 17.8 atm in the presence of 0.05 M-maleic acid-NaOH at pH 5.8. Protoplasts were released through ruptures in the wall, initially at the apices, but later also from older parts of the hyphae. I N 'l- R 0 D U C T I0 N There are a number of studies on the liberation of protoplasts from filamentous fungi (reviewed by Strunk, 1970). The organisms described represent all the major groups of fungi, but only one study concerns a basidiomycete (Polysficrus twsicolor) (Strunk, 1969). In general, quantitative data were not given on the optimal conditions for the liberation of protoplasts. Schizophyllum commune has been used extensively for studies on differentiation and morphogenesis (Niederpruem & Wessels, 1969 ; Wessels, 1971) and for this reason we investi- gated the conditions which favour a reproducible and efficient release of protoplasts from the hyphae of this basidiomycete. The availability of protoplasts may be very useful for further morphological, biochemical and genetic studies with this organism. We report here the effective use of an enzyme preparation derived from Trichodt?rma iyiride for the release of protoplasts from the hyphae of S. commune. Organisms. The strains of Schizop/z~~lluni commune used were 1 he homokaryotic strain 699 obtained from Dr J. R. Raper (Harvard University, Cambridge, Massachusetts, U.S.A.) and a dikaryotic strain KS derived from the KNIEP stock (Wessels, 1965). Trichoderma Ijiride (CBS 354.33) was obtained from the Centraalbureau voor Schimmelcultures, Baarn, The Net herlands. Lytic enzjw7e.s. To obtain enzymes lytic towards walls of Schiz-ophylhm commune, Tricho- dernia tiride was grown in a liquid medium containing glucose (0.3 %), S. commune hyphal walls (0.5 %) and mineral components (Mandels, Parrish & Reese, I 962). The cultures were incubated for 8 days at 25 "C on a New Brunswick gyratory shaker (225 rev./min). Hyphal walls were prepared from the mycelium of S. commune stock 1i8 grown in shaken cultures