An efficient transformation system for the fungus Trichoderma longibrachiatum has been developed. Transformation was obtained both by electroporation and polyethyleneglycol treatment, using a plasmid carrying the Escherichia coli hygromycin B phosphotransferase gene as a dominant selectable marker. The transformation frequency was 0.5 to 5 transformants /mu g plasmid DNA. Transformation normally occurred by tandem integration of the transforming DNA. A high percentage of the transformants were mitotically unstable. The efficiency of co-transformation was very high (around 90%), and several co-transformants containing multiple copies of the egl1 gene encoding a P(1,4)-endoglucanase were obtained. Some of them secrete increased levels of endoglucanase to the culture medium. In addition, the E. coli lacZ gene was expressed in an active form under control of the Aspergillus nidulans gpdA gene promoter.