To obtain cells with high dibenzothiophene (DBT) degrading activity, two phase fed-batch culture of Rhodococcus erythropolis KA 2-5-1 was carried out, which consisted of growth phase in the medium containing sulfate ion as a sulfur source and induction phase for expression of DBT degrading enzyme by DBT addition. Cell concentration of 24.3 g/L was obtained after 30 h culture and the maximum specific degrading activity of 18.2 mmol/kg-dry cells/h was achieved at 10 h after induction. In order to simplify the system and lower an operational cost, next, in situ induction of the enzyme activity was conducted by means of sulfur limitation since the strain can only be induced under sulfur starvation. The strain was cultivated in fed-batch mode in the modified A medium containing 96 mg/L of sulfate ion. At 22 h, sulfate concentration decreased below 10 mg/L and approximately 10 g/L of cell concentration was obtained. When 125 mg/L of DBT was added at 22 h, the enzyme activity increased rapidly and the maximum specific activity of 14.3 mmol/kg-dry cells/h was obtained at 28 h. However, the DBT degrading activity decreased rapidly thereafter. From the assay of remaining concentration of medium components in the culture broth, it was found that Fe and Mg ions were exhausted at the end of cultivation. Fed-batch culture with feeding of concentrated solution of Fe and Mg ions was then conducted. After 23 h, 125 mg/L of DBT was added, and the enzyme activity increased rapidly. In this case, the activity did not decrease, and the maximum specific degrading activity of 18.5 mmol/kg-dry cells/h and cell concentration of 41.7 g/L were obtained at 34 h.