The sulfate repression of desulfurization (Dsz) phenotype represents a major barrier to the mass production of desulfurizing resting cells. This repression can be avoided by replacing sulfate with dibenzothiophene (DBT) as the main substrate for the 4S pathway. However, mass production of biocatalyst using DBT is impractical because of its high price, low water solubility, and growth inhibition by 2-hydroxybiphenyl (2-HBP), which is the end product of the 4S pathway. In this work, the results showed that readily bioavailable sulfur compounds led to repression of the desulfurization activity of Gordonia alkanivorans RIPI90A. However, the Dsz phenotype was expressed through the 4S pathway in the presence of DMSO as the sulfur source for growth. Resting cells grown on DMSO were more active than the resting cells grown on DBT. The growth rate of strain RIPI90A on DMSO was higher than when DBT was used as the sole sulfur source. DMSO concentration significantly influenced the growth pattern of the strain, and the highest growth rate was observed at a concentration of 200 microg ml(-1). Above this concentration, the growth rate gradually decreased. DBT was found to induce the Dsz phenotype, with no observed lag period, in cells grown on DMSO as the sole sulfur source. Prior to induction, the specific activity was detected as 1.4 micromol 2-HBP (g dry cell weight)(-1) h(-1), and following incubation (5 h) the highest specific activity was observed as 5.11 micromol 2-HBP (g dry cell weight)(-1 )h(-1). This study identified that resting cells can be prepared in a two-step process. First, resting cells can be produced using DMSO as the sulfur source for growth; in the second step, improvements to their desulfurizing activity can be made using DBT as an inducer. DMSO is recommended as an appropriate sulfur source for the mass production of G. alkanivorans RIPI90A.