Protoplasts were obtained fromHyphoderma roseum(Fries) andCunninghamella elegans(Lendner), fungi capable of steroid 11-hydroxylation. The lytic enzyme preparation was derived fromTrichoderma virideCBS 354-33. Homogeneous protoplast suspension, free of mycelial debris and cell wall fragments, transformed cortexolone and 6α-fluorocortexolone-16,17-acetonide to the same products as the intact mycelium of the microorganisms. Liberation of protoplasts and their stabilizaiton during steroid transformation was the most effective in 0.8 MMgSO4; still, this compound impaired steroid hydroxylation. Consequently, the concentration of the transformation product formed was nearly the same as in sucrose, mannitol, and sorbitol, compounds which caused no inhibition but which were less effective stabilizers.