The substrate specificity of Mycobacterium sp. G3 with desulfurization activity against dibenzothiophene (DBT) was investigated. Desulfurization reactions were carried out using a concentrated cell suspension of G3. The conversion from 4,6-dipropyl DBT, one of the sulfur-containing compounds that is difficult to desulfurize in diesel oil, to 2-hydroxy-3,3'-dipropylbiphenyl as an end-product of the desulfurization reaction was found in the water reaction system and in the oil/water two-phase reaction system. 4,6-Dibutyl DBT and 4,6-dipentyl DBT were metabolized to he hvdroxybiphenyl form via the sulfone form in the water reaction system. These results indicate that G3 has high membrane permeability and superior substrate specificity for high molecular weight alkyl DBTs, which are represented by 4,6-dipentyl DBT as C10-DBT. Furthermore, G3 could desulfurize diesel oil, and the sulfur concentration was decreased from 116 mg l(-1) to 48 mg l(-1) within 24 h.