Sphaeroplasts from several genetically marked strains of Trichoderma reesei were readily prepared through enzymatic hydrolysis of mycelial suspensions utilizing Driselase, a commercially available lytic enzyme preparation. Sphaeroplasts were released from the apical tips of hyphae after 90 min exposure to the enzyme and, with longer treatments, from other regions of the hyphae. The efficiency of sphaeroplast formation was dependent upon a number of factors. Young mycelium was far more susceptible to lysis than older hyphae. Additionally, the yields of sphaeroplasts were directly proportional to the mycelial concentration within the range tested (0.7–7.0 mg dry weight of mycelium). Ammonium sulfate (0.6 M) was the most effective osmotic support in relation to sphaeroplast stability during their release from the mycelium and subsequent purification. The most prominent enzymatic activities of Driselase were endo-β-(1→4)-glucanase, β-glucosidase and β-(1→3)-glucanase. No chitinase activity was detected. The combined application of Driselase and chitinase (Streptomyces griseus) to mycelial suspensions resulted in increased sphaeroplast yields from young (18 h) as well as older (24 h) mycelia. The parameters defined here allow the rapid (<6h), inexpensive production of 107 sphaeroplasts/ml. Regeneration of T. reesei sphaeroplasts proceeded by the production of chains of sphaeroplast-like cells followed by true hyphal formation. The frequency of regeneration to mycelial form was dependent upon the length of exposure of mycelium to the lytic enzyme. Less than 1% of the sphaeroplasts prepared from 24 h hydrolytic treatment of mycelia regenerate to form colonies while virtually 100% of the viable sphaeroplasts prepared from 6 h treatment form colonies in regeneration medium.