Protoplasts were isolated from Trichoderma reesei strain PTr2 using Lysing enzymes (Sigma Chemicals Co., USA) with 0.6 M KCl as osmotic stabilizer. Intra-strain protoplast fusion has been carried out using 40% polyethylene glycol with STC (sorbitol, Tris-HCl, CaCl2) buffer. The fused protoplasts of T. reesei have been regenerated on carboxymethyl cellulose agar (CMCA) selective medium and 15 fusants were selected for further studies. Most of the fusants exhibited fast mycelial growth and abundant sporulation on PDA as compared to non-fusants and parent strains. Prominent clearing zones that appeared around the mycelial growth of most of the fusant progenies when grown on CMCA indicated the secretion of high level extracellular carboxymethyl cellulase (CMCase) than the non-fusants and parent. High CMCase activity was estimated with 80% of the fusants and more than two-fold increase in enzyme activity was recorded with two fusants, SFTr2 and SFTr3 as compared to the parent strain PTr2. Results of the present study demonstrated the scope and significance of the protoplast fusion technique, which can be used to develop superior hybrid strains of filamentous fungi that lack inherent sexual reproduction. (c) 2005 Elsevier Inc. All rights reserved.