Protoplasts from two auxotrophic mutants ofTrichoderma harzianumRifai (ATCC 32173), obtained from young thalli following cell wall digestion by NovoZym 234, were fused in 33% PEG suspended in 10 mM Tris-HCl and 10 mM CaCl2, pH 7.5. The frequency of fusion between lysine- and arginine-requiring auxotrophs resulting in prototrophic strains was about 5%. These prototrophic strains were classified into parental and nonparental types. Colonies developed from single conidia of the nonparental phenotype exhibited prototrophic parental or recombinant phenotypes. The ability of both prototrophic and parental strains to overgrow the soil-borne pathogenic fungiRhizoctonia solani,Sclerotium rolfsii, andPythium aphanidermatumin dual cultures was used to evaluate their antagonistic capability. The antagonistic abilities of the prototrophic strains were found to vary with each pathogenic fungus. The prototrophic strain A2 overgrew all the pathogenic fungi more rapidly than the parental strains. Strain A2 effectively controlledRhizoctoniadamping-off of cotton seedlings, in the greenhouse, when compared with the parental strains. Protoplast fusion appears to be a useful tool for combining desirable traits from parental strains to produce improved biocontrol strains.Key words:Trichoderma harzianum, biocontrol, protoplast fusion.