A Thermus thermophilus selector strain for production of thermostable and thermoactive -galactosidase was constructed. For this purpose, the native -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 (-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 -galactosidase. Moreover, the AgaT strain had to be Km^s for establishment of the plasmids containing -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 -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.