Anaerobic biodegradation of toluene and ethylbenzene is of environmental concern and biochemical interest due to toxicity and novel reactions, respectively. The denitrifying strain EbN1 is unique in anaerobically degrading both alkylbenzenes via different pathways which converge at benzoyl coenzyme A. The organization of genes involved in both pathways was only recently determined for strain EbN1. In the present study, global expression analysis (DNA microarray and proteomics) indicated involvement of several thus-far-unknown proteins in the degradation of both alkylbenzenes. For example, orf68 and orf57, framing the ebd operon, are implicated in ethylbenzene degradation, and the ebA1932 and ebA1936 genes, located 7.2 kb upstream of the bbs operon, are implicated in toluene degradation. In addition, expression studies were now possible on the level of the complete pathways. Growth experiments demonstrated that degradative capacities for toluene and ethylbenzene could be simultaneously induced, regardless of the substrate used for adaptation. Regulation was studied at the RNA (real-time reverse transcription-PCR and DNA microarray) and protein (two-dimensional-difference gel electrophoresis) level by using cells adapted to anaerobic growth with benzoate, toluene, ethylbenzene, or a mixture of toluene and ethylbenzene. Expression of the two toluene-related operons (bss and bbs) was specifically induced in toluene-adapted cells. In contrast, genes involved in anaerobic ethylbenzene degradation were induced in ethylbenzene- and toluene-adapted cells, suggesting that toluene may act as a gratuitous inducer. In agreement with the predicted sequential regulation of the ethylbenzene pathway, Ebd proteins (encoding subunits of ethylbenzene dehydrogenase) were formed in ethylbenzene- but not in acetophenone-adapted cells, while Apc proteins (subunits of predicted acetophenone carboxylase) were formed under both conditions.