In Hypocrea jecorina (anamorph: Trichoderma reesei) multiple gene deletions are limited by the number of readily available selection markers. We have therefore constructed a blaster cassette which enables successive gene knock-outs in H. jecorina. This 3.5 kb pyr4 blaster cassette contains the H. jecorina pyr4 marker gene encoding orotidine-5'-monophosphate (OMP) decarboxylase flanked by two direct repeats of the Streptoalloteichus hindustanus bleomycin gene (Sh ble), which facilitate the excision of the blaster cassette by homologous recombination after each round of deletion. Functionality of this pyr4 blaster cassette was demonstrated by deletion of the glk1 encoding glucokinase and hxk1 encoding hexokinase. 1.4-1.8 kb of the non-coding flanking regions of both target genes were cloned into the respective blaster cassettes and transformation of a pyr4 negative H. jecorina strain with the two cassettes resulted in 10-13% of the transformants in the deletion of one of the two kinase genes. For excision of the pyr4 blaster cassettes, Deltaglk1 strains were selected for growth in the presence of 5-fluoroorotic acid. Recombination between the two Sh ble elements resulted in uridine auxotrophic strains which retained their respective glucokinase negative phenotype. Subsequent transformation of one of these auxotrophic Deltaglk1 strains with the hexokinase blaster cassette resulted in pyr4 prototrophic strains deleted in both glk1 and hxk1. Deltaglk1 strains showed reduced growth on d-glucose and d-fructose whereas Deltahxkl strains showed reduced compact growth on d-glucose but were unable to grow on d-fructose as carbon source. The double Deltaglk1Deltahxk1 deletion strain was completely unable to grow on either d-glucose or d-fructose.