Recombinant Mycobacterium sp. strain MR65 harboring dszABCD genes was used to desulfurize alkyl dibenzothiophenes (C-x-DBTs) in n-hexadecane. The specific desulfurization activity for 2,4,6,8-tetraethyl DBT (C-8-DBT) by DszC enzyme was about twice that for 4,6-dipropyl DBT (C-6-DBT). However, the degradation rate of 2,4,6,8-tetraethyl DBT in n-hexadecane by resting cells of strain MR65 was only about 40% of that of 4,6-dipropyl DBT. These results indicated that the desulfurization ability for C-x-DBTs by resting cells depends on carbon number substituted at positions 4 and 6 and that the rate-limiting step in the desulfurization reaction of highly alkylated C-x-DBTs is the transfer process from the oil phase into the cell.