Alternaria alternata is well known as a producer of tentoxin as well as some other phytotoxic substances. A new method to prepare protoplasts from Alternaria alternata, suitable for many purposes, was developed. By use of a mixture of lytic enzymes from Helix pomatia, and Trichoderma harzianum with the commercial preparation "novozym," it was possible to prepare protoplasts from all stages of fungal development, including the tentoxin production phase. Optimal incubation conditions led to the conversion of 1 g (wet wt) mycelial cells into 2.3-2.5 x 10(7) protoplasts within 3-6 h. Submerged as well as surface-grown mycelia were suitable. Optimal stabilization of protoplasts was obtained in 0.8M KCl. The protoplasts were used for both mutagenic treatment and physiological studies. UV irradiation of protoplasts resulted in formation of hyperproducing mutants. Protoplasts were able to form tentoxin. The biosynthetic activity of protoplasts from surface-grown mycelium was 40% that of intact mycelia. Although intact submerged myclia did not synthesize tentoxin, protoplasts of both types of mycelia produced this toxin, indeed protoplasts from submerged mycelia were even more active than those from surface mycelia. Neither oxygen tension nor mechanical stress during the shaking culture is the reason for the lack of tentoxin production by intact submerged mycelial pellets. Since tentoxin-synthesizing enzymes were apparently present in both mycelial types, it is probable that metabolites or lytic products in the pellets inhibit tentoxin-forming enzymes under submerged conditions.