Protoplast fusion of strains derived from Trichoderma reesei QM9414 and QM9136 and the segregation of the resulting fusants were studied. Combinations of protoplasts prepared from young conidia with double amino acid requirements, one of which was a common requirement and the other uncommon, were fused in the presence of polyethylene glycol 6000. Fusants were selected as regenerant colonies requiring only the commonly deficient amino acid. The frequency of fusion was 0.9 x 10-4 to 4.0 x 10-4 for the starting conidia and 3.0 x 10-2 to 4.9 x 10-2 for the regenerated protoplasts, which was significantly higher than the expected reversion frequencies by mutation. Conidia generated on the fusant colonies showed diverse phenotypes, i.e., parental types (40 to 80%) and nonparental types (20 to 60%). Colonies developed from single conidia of the nonparental phenotype contained special spots called "knobs" that have a higher density of mycelia. The phenotype of the knobs was again varied among prototrophs, parental types, and recombinant types; and their traits were inherited stably. The phenotype of the mycelia in the nonknob part was essentially the same as that of the original conidia and again formed knobs in colonies upon transfer of a piece of mycelia to a fresh medium. The conidial DNA content of the knob clone was almost the same as that of the parents, but that of the fusants was 1.2 to 2.0 times higher than that of the parents. From these results, we conclude that knobs are the segregants from the fusants. One knob clone showed twice the carboxymethyl cellulose hydrolyzing activity of the parents, suggesting the possibility of breeding T. reesei cells by the protoplast fusion technique.