Context The medial frontal cortex (MFC), including the dorsal anterior cingulate and the supplementary motor area, is critical for adaptive and inhibitory control of behavior. Abnormally high MFC activity has been a consistent finding in functional neuroimaging studies of obsessive-compulsive disorder (OCD). However, the precise regions and the neural alterations associated with this abnormality remain unclear. Objective To examine the functional and biochemical properties of the MFC in patients with OCD. Design Cross-sectional study combining volume-localized proton magnetic resonance spectroscopy and functional magnetic resonance imaging with a task encompassing inhibitory control processes (the Multi-Source Interference Task) designed to activate the MFC. Setting Healthy control participants and OCD patients recruited from the general community. Participants Nineteen OCD patients (10 males and 9 females) and 19 age-, sex-, education-, and intelligence-matched control participants recruited from the general community. Main Outcome Measures Psychometric measures of symptom severity, Multi-Source Interference Task behavioral performance, blood oxygen level–dependent activation, and proton magnetic resonance spectroscopy brain metabolite concentrations. Results Multi-Source Interference Task behavioral performance did not differ between OCD patients and control subjects. Reaction time interference and response errors were correlated with blood oxygen level–dependent activation in the dorsal anterior cingulate region in both groups. Compared with controls, OCD patients had greater relative activation of the supplementary motor area and deactivation of the rostral anterior cingulate during high- vs low-conflict (incongruent > congruent) trials. Patients with OCD also showed reduced levels of neuronal N-acetylaspartate in the dorsal anterior cingulate region, which was negatively correlated with their blood oxygen level–dependent activation of the region. Conclusions Hyperactivation of the MFC during high- vs low-conflict conditions in patients with OCD may be a compensatory response to a neuronal abnormality in the region. This relationship may partly explain the nature of inhibitory control deficits that are frequently seen in this group and may serve as a focus of future treatment studies.