ABSTRACTSeveral Plasmids with different promoter sequences, all containing theHygBgene that confers resistance to the antibiotic hygromycin B, were evaluated for their ability to transformTrichoderma viridestrain T105-227 Nie-. Plasmid pH1B, which contains a promoter sequence fromCochliobolus heterostrophus, was found to give the largest number of putative transformants. Transformation ofT. harzianumstrains T12-2 His- and T95-1 Lys- was attempted, and the time at which transformed protoplasts were exposed to hygromycin B was found to be critical. If hygromycin B was added before protoplasts had regenerated for at least 6 h, no transformants were obtained. However, if regeneration proceeded for more than 12 h, many nontransformed protoplasts formed thalli. Putative transformants were heterokaryotic. Even if thalli were grown in the presence of hygromycin B, the number of transformed nuclei was low; only 0.5 to 0.8% of conidia from putative transformants were capable of producing thalli in the presence of hygromycin B. Subcultures derived from single hygromycin B-resistant conidia were stable. Southern analysis indicated that the plasmid was integrated into the genome in either tandem repeat pattern or was single copy insertion.