In contrast to mechanical disruption of Alternaria alternata mycelia, disintegration of protoplasts by osmotic shock made it possible for the first time to develop a cell-free system that produces tentoxin without significant loss of enzyme activity. After separation of groups of cell organelles by fractional centrifugation, the main activity of tentoxin synthesis was detectable in the membrane/microsome fraction and in the plasma fraction. The proteins in the plasma seem not to be soluble. They are probably combined with lipids or small bits of membrane debris. For this reason, these proteins cannot be removed by ultracentrifugation. On the other hand, it is possible to precipitate the enzyme system with 2% NaCl. Disruption of cellular substructures (membranes?) by mechanical disintegration or by various detergents led to a rapid loss of enzyme activity. The tentoxin-synthesizing enzyme is probably localized at or in membranes. De novo synthesis of tentoxin in a cell-free system devoid of ribosomes from radioactively labeled amino acids shows that tentoxin is not a virus product.