ABSTRACTHigh yields (approx. 108/ml) of protoplasts of high purity (>99%) can be obtained from young thalli of strains T12 and T95 ofTrichoderma harzianumby digestion of cell walls with NovoZym 234. Protoplasts derived from mycelium or immature conidia contained 2–12 nuclei. After resuspension in 0.6 M sorbitol, 10 mM CaCl2, and 10 mM Tris-HCl at pH 7.5 (STC) about 10% of the protoplasts regenerated on osmotically stabilized medium. Protoplasts fused by a step-wise addition of polyethylene glycol (PEG) also regenerated readily. Fusion between lysine-requiring and histidine-requiring auxotrophs of strain T95 gave rise to about 10% as many colonies on basal medium (BM) as on BM amended with histidine and lysine (BM + HL). Conidia from these prototrophic colonies were nearly all auxotrophic. Approximately equal numbers required histidine or lysine. These results indicate that the prototrophic progeny of this fusion were balanced heterokaryons with the parental nuclei present in equal numbers. Conversely, fusion between T12 his and T 95 lys gave rise to unbalanced heterokaryons with T12 predominating. The frequency of prototroph formation was less than 0.01% and the original isolates grew very slowly. These expressed only the isozyme phenotype of the T12 parent at four loci but the T95 genome was detected in sectors and single-spore isolates. Subsequently, various non-parental progeny were recovered. Some were of the T95 isozyme phenotype, while others were of the T12 phenotype. None showed isozyme patterns indicative of expression of both genomes. Growth rates ranged from greater than that of either wild type parent on any medium to very slow growth rates even on permissive media. Morphologies ranged from wrinkled, brown, nearly asporulent types recognizable asTrichodermaonly by isozyme phenotype to ones similar to the wild-type parental strains. Many nonparental strains remained unchanged when propagated from single conidia, indicating that these nonparentals were homokaryotic. Others yielded a variety of parental and nonparental single-spore isolates, indicating complex heterokaryosis.