Various carbon and sulfur sources on the growth and desulfurization activity of Mycobacterium strain G3, which is a dibenzothiophene (DBT)-degrading microorganism, were studied. Ethanol, glucose or glycerol as the sole carbon source and MgSO4, taurine or dimethyl sulfoxide (DMSO) as the sole sulfur source were suitable for the growth. In addition, desulfurization activity was expressed in medium containing taurine, MgSO4 or DMSO at 0.1 mM, when 217 mM ethanol was used as the sole carbon source. The highest desulfurization activity was in the stationary phase cells after 5 days' growth, rather than those harvested during active growth, when Mycobacterium G3 was cultivated in medium containing 217 mM ethanol and 0.1 mM MgSO4. Thus alternative sulfur sources to DBT can be used for the cultivation of this desulfurizing microorganism.