The denitrifying strain T1, identified as Thauera aromatica, is able to grow with toluene serving as its sole carbon source. Previous work identified two genes, tutD and tutE, that are involved in toluene metabolism. Two small open reading frames, tutF and tutG, which may also play a role in toluene metabolism, were also identified. The present work examines the transcriptional organization and regulation of these toluene utilization genes. Northern analysis indicates that the four genes are organized into two operons, tutE and tutFDG, and that both operons are regulated in response to toluene. Primer extension analysis has identified major transcriptional start sites located 177 bp upstream of the tutE translational start and 76 bp upstream of the tutF translational start. Furthermore, a fifth gene, tutH, has been identified immediately downstream of tutG. It is transcribed from the same start site as tutFDG and is predicted to code for a 286-amino-acid protein with a calculated molecular mass of about 31,800 Da. The TutH protein is predicted to have an ATP/GTP binding domain and is similar to the NorQ/NirQ family of proteins.