To construct a genetically stable strain of Serratia marcescens for d-biotin production, we integrated the mutated biotin-biosynthetic (bio) genes into the chromosome of a d-biotin-producing strain of S. marcescens Sr41. Temperature-sensitive suicide vectors consisting of the Tn5 element were used as the integration tool. Extra bio genes carried between the Tn5 termini were integrated into the chromosome of strain ETA23, a d-biotin-producing mutant. Resultant integrants produced 120 mg of d-biotin per l of a medium containing sucrose and urea whereas ETA23 produced d-biotin at 55 mg/l in the same medium. The integrated bio genes were stably maintained on the chromosome of ETA23 for 100 generations.