Previously we reported that double disruption of the proteinase genes (tppA and pepE) improved heterologous protein production by Aspergillus oryzae (Jin et al. Appl Microbiol Biotechnol 76:1059-1068, 2007). Since A. oryzae has 134 protease genes, the number of auxotrophy in a single host is limited for multiple disruptions of many protease genes. In order to rapidly perform multiple gene disruptions in A. oryzae, we generated the marker recycling system in highly efficient gene-targeting background. A. oryzae ligD gene homologous to Neurospora crassa mus-53 gene involved in nonhomologous chromosomal integration was disrupted, followed by disruption of the pyrG gene for uridine/uracil auxotroph. We further performed successive rounds of gene disruption (tppA and pepE) by the pyrG marker with high gene-targeting efficiency allowed by the Delta ligD background. After each disruption process the pyrG marker was excised by the direct repeats consisting of similar to 300 bp upstream flanking region of the target gene, resulting in no residual ectopic/foreign DNA fragments in the genome. Consequently, we succeeded to breed the double proteinase gene disruptant (Delta tppA Delta pepE) applicable to further sequential gene disruptions in A. oryzae.