Benzene (C 6H6) is the simplest member of the aromatic hydrocarbon group of chemical compounds. Minute amounts of benzene are naturally released into the environment during volcanic eruptions and forest fires. This extremely stable aromatic compound is also an important industrial chemical and is an integral component of many petroleum products. In fact, benzene is amongst the top 20 in production volume for chemicals produced in the United States. Therefore, it is not surprising that the major reason for environmental contamination through benzene is by anthropogenic sources. Benzene is relatively soluble in water and migrates very quickly in the soil after its entry. The Environmental Protection Agency (EPA) has classified benzene as a Class A carcinogen. Microorganisms play an integral role in the natural attenuation of benzene from the environment. Biodegradation of benzene by oxidation can occur under aerobic, anaerobic and microaerophilic conditions. Biooxidation of benzene under aerobic conditions is well-studied. However, oxygen is scarce in contaminated subsurface environments, and after the aerobic breakdown of benzene, oxygen is quickly depleted from the most heavily contaminated regions leading to the development of extensive anaerobic zones. As a result, there is increased focus on anaerobic benzene degradation as a potential bioremediation technique in anoxic subsurface environments. In aerobic and microaerophilic environments, monooxygenase and dioxygenase enzyme systems have been established to be involved in the breakdown of the benzene ring. However, the genes and enzymes involved in anaerobic benzene oxidation pathway are still unknown. In the present study, Dechloromonas aromatica strain RCB, capable of benzene oxidation with nitrate as the electron acceptor, was used as a model system to investigate the initial steps of the anaerobic benzene oxidation pathway. Strain RCB is capable of completely mineralizing benzene to carbon dioxide in denitrifying conditions. RNA-arbitrarily primed polymerase chain reaction (RAP-PCR), a differential gene expression technique used to randomly reverse-transcribe RNA into cDNA, was conducted to identify genes exclusively expressed during nitrate-dependent benzene oxidation. A total of seven genes were identified as differentially expressed in the presence of benzene using the RAP-PCR approach. Four differentially expressed genes were confirmed by a second method, semiquantitative reverse transcriptase PCR (RT-PCR). Microarray analysis was the second expression analysis technique conducted to identify genes expressed during benzene-oxidizing conditions. Based on fold induction and potential function, six genes were selected from the microarray data and their differential expression was confirmed by using semiquantitative RT-PCR. Interestingly, Daro1556, encoding a hypothetical protein, was identified by both RAP-PCR and microarray analysis. In order to verify the functions of the genes (selected from RAP-PCR and microarray analysis) in nitrate-dependent benzene oxidation, six deletion mutants were constructed in which the target gene was replaced by a tetracycline cassette. The correct insertion of the tetracycline cassette in the mutant genome was confirmed by PCR and Southern blotting. Microarray results were further analyzed by using an unsupervised clustering approach, k-means. A couple of genes (Daro1358 and Daro1359) obtained from cluster analysis were also verified by semiquantitative RT-PCR. These two genes, part of the same operon, encode a two-component monooxygenase system, which is a member of the Rieske non-heme iron aromatic ring-hydroxylating oxygenase family of proteins. In the present investigation, for the first time, involvement of a monooxygenase system (Daro1358 and Daro1359) during benzene oxidation with nitrate reduction was observed. Based on the results obtained from k-means cluster analysis, a model was hypothesized for anaerobic benzene oxidation with nitrate as the electron acceptor in Dechloromonas aromatica strain RCB.