Converging neuropsychological and functional neuroimaging evidence indicates that the dorsal anterior cingulate cortex (dACC) is dysfunctional in drug-addicted populations. Few studies, however, have investigated the biochemical and physiological properties of the dACC in such populations. We used proton magnetic resonance spectroscopy ((1)H-MRS) together with functional magnetic resonance imaging (fMRI) to probe dACC biochemistry and physiological activity during performance of a behavioural control task in 24 opiate-dependent individuals (maintained on a stable dose of methadone or buprenorphine at the time of study) and 24 age, gender, intelligence and performance-matched healthy subjects. While both groups activated the dACC to comparable levels, the opiate-using group displayed relatively increased task-related activation of frontal, parietal and cerebellar regions, as well as reduced concentrations of dACC N-acetylaspartate and glutamate/glutamine. In addition, the opiate-using group failed to show the expected correlations between dACC activation and behavioural measures of cognitive control. These findings suggest that the dACC is biochemically and physiologically abnormal in long-term opiate-dependent individuals. Furthermore, opiate addicts required increased, perhaps compensatory, involvement of the fronto-parietal and cerebellar behavioural regulation network to achieve normal levels of task performance/behavioural control. These neurobiological findings may partly underpin key addiction-related phenomena, such as poor inhibitory control of drug-related behaviour in the face of adverse consequences, and may be of relevance to the design of future treatment studies.