Starch and maltooligosaccharides such as maltose and maltotriose induce the production of amylolytic enzymes including alpha-amylase in Aspergillus oryzae. A transcriptional activator gene amyR, required for maltose induction of amylolytic enzymes, has been cloned and characterized. The amyR gene deletion mutant showed significantly poor growth on starch medium but normal growth on maltose medium. This indicated the existence of another maltose-utilizing system, whose expression might not be controlled by amyR. We have identified a gene cluster homologous to the MAL cluster of Saccharomyces cerevisiae in the A. oryzae genome. The cluster consists of a MAL61 homolog (designated malP), a MAL62 homolog (designated malT), and a MAL63 homolog (designated malR). Overexpression of malT in A. oryzae resulted in a significant increase in intracellular alpha-glucosidase activity, and that of malP allowed S. cerevisiaemal61Delta to grow on maltose. The expression of both malP and malT genes was highly up-regulated in the presence of maltose, but malR expressed constitutively irrespective of carbon sources. Disruption of malR resulted in the loss of malP and malT expression and thus in restricted growth on maltose medium. In addition, a malP disruptant showed a significantly reduced expression of malT and malR and exhibited a growth defect on maltose similar to the malR disruptant. These results suggest that the MAL cluster of A. oryzae is responsible for the assimilation of maltose in A. oryzae.