A Thermus thermophilus selector strain for production of thermostable and thermoactive alpha-galactosidase was constructed. For this purpose, the native alpha-galactosidase gene (agaT) of T. thermophilus TH125 was inactivated to prevent background activity. In our first attempt, insertional mutagenesis of agaT by using a cassette carrying a kanamycin resistance gene led to bacterial inability to utilize melibiose (alpha-galactoside) and galactose as sole carbohydrate sources due to a polar effect of the insertional inactivation. A Gal(+) phenotype was assumed to be essential for growth on melibiose. In a Gal(-) background, accumulation of galactose or its metabolite derivatives produced from melibiose hydrolysis could interfere with the growth of the host strain harboring recombinant alpha-galactosidase. Moreover, the AgaT(-) strain had to be Km(s) for establishment of the plasmids containing alpha-galactosidase genes and the kanamycin resistance marker. Therefore, a suitable selector strain (AgaT(-) Gal(+) Km(s)) was generated by applying integration mutagenesis in combination with phenotypic selection. To produce heterologous alpha-galactosidase in T. thermophilus, the isogenes agaA and agaB of Bacillus stearothermophilus KVE36 were cloned into an Escherichia coli-Thermus shuttle vector. The region containing the E. coli plasmid sequence (pUC-derived vector) was deleted before transformation of T. thermophilus with the recombinant plasmids. As a result, transformation efficiency and plasmid stability were improved. However, growth on minimal agar medium containing melibiose was achieved only following random selection of the clones carrying a plasmid-based mutation that had promoted a higher copy number and greater stability of the plasmid.