Work with Rhizopus oryzae previously suggested that this filamentous fungus only had one glucoamylase gene. We demonstrate in this study that some R. oryzae strains contain multiple glucoamylase genes with differential regulation. The existence of the two unique, amy genes in R. oryzae NRRL 395, a Type-I strain, was confirmed by Southern blot analysis, restriction mapping, and sequencing. Sequence analysis of the amy genomic clones reveals extensive homology in the coding region, as well as the flanking ends. A notable difference is the amyB gene does not encode a starch binding domain and also contains a 24 bp insertion in the open reading frame that remains in the cDNA. In addition, isolation of the amyA and amyB cDNA demonstrates that the single common intron is spliced in an identical manner. The deduced amino acid sequence of the amy genes shares 91% homology, excluding the starch binding domain. The putative glucoamylase enzymes contain similar signal sequences, as well as conserved amino acid sequence regions found in family 15 glycoside hydrolases. Transcriptional analyses demonstrate that both amy genes are highly expressed on solid medium with low water activity. However, amyB demonstrates little or no expression in shake flask cultures with various carbon sources. Recombinant expression of AmyB in Pichia pastoris resulted in production of full-length protein, but no detectable glucoamylase activity. (c) 2006 Elsevier Inc. All rights reserved.