Rhodococcus opacus B-4 is a benzene-tolerant bacterium which was isolated from a gasoline-contaminated soil sample. We previously demonstrated that this organism was able to survive and exhibit biocatalytic activity in anhydrous organic solvents for at least 5 d. In the present study, we cloned the alkB1 and alkB2 genes encoding alkane hydroxylases from R. opacus B-4. Heterologous expression of the alkB1 and alkB2 genes in Escherichia coli JM 109 showed that they encode functional alkane hydroxylases with a substrate range of C-5-C-16. Promoters of the alkB1 and alkB2 genes, designated P-alkB1 and P-alkB2, respectively, were examined for activity in anhydrous his (2-ethylhexyl) phthalate (BEHP) containing C-5-C-16 n-alkanes. Two recombinant plasmids, pP(alkB1)EGFP and pP(alkB2)EGFP, were constructed by inserting the egfp gene downstream of P-alkB1 and P-alkB2, respectively and transformed into R. opacus B-4. Resting cells of R. opacus B-4 (pP(alkB1)EGFP) showed greater levels of EGFP fluorescence in anhydrous BEHP than in 0.85% NaCl, when C-8-C-16 n-alkanes were supplied as an inducer. Furthermore, n-alkane inducibility of P-alkB1 activity in anhydrous BEHP was noticeably different from that in 0.85% NaCl. This paper presents the first evidence that bacteria can express their genes in essentially anhydrous organic solvents.