The glaA gene encoding glucoamylase I (GAI) of Aspergillus awamori var. kawachi was successfully expressed in cytoplasm of Saccharomyces cerevisiae, using two cytoplasmic linear plasmids, pGKL1 and pGKL2. In order to integrate the glaA gene, two pGKL1-derived plasmids, designated pKTF951 and pKTF952, were constructed. The glaA gene was placed downstream of the ORF2 promoter (UCS2) between ORF1 and ORF3 of pGKL1 to obtain pKTF951. pKTF952 contained an additional ORF5 promoter (UCS5) from pGKL2, connected downstream of the UCS2 in pKTF951. pKTF951 and pKTF952 were respectively transformed to S. cerevisiae carrying the original pGKL1 and pGKL2. Southern analysis revealed that in vivo homologous recombination took between the indigenous pGKL1 and the recombinant pKTF951 or pKTF952 within the common ORF1 and ORF3 regions, which resulted in the replacement of ORF2 by the glaA gene In pGKL1. Transformants carrying the resultant linear recombinant plasmids, pNS951 or pNS952, produced GAI. S. cerevisiae (pNS952) secreted 2.6-fold more GAI than S. cerevisiae (pNS951).