J. Neurochem.(2010)114, 1745–1755.AbstractCatechol‐O‐methyltransferase (COMT) plays an active role in the metabolism of dopamine (DA) in the prefrontal cortex (PFC). Because of low levels of dopamine transporter (DAT), it is proposed that the majority of released DA is taken up by either norepinephrine transporter (NET) and subsequently metabolized by monoamine oxidize (MAO) or by uptake2(to glial cells and post‐synaptic neurons) and metabolized by COMT. However, a comprehensivein vivostudy of rating the mechanisms involved in DA clearance in the PFC has not been done. Here, we employ two types of microdialysis to study these pathways using DAT, NET and MAO blockers in conscious mice, with or withoutComtgene disruption. In quantitative no‐net‐flux microdialysis, DA levels were increased by 60% in the PFC of COMT‐knockout (ko) mice, but not in the striatum and nucleus accumbens. In conventional microdialysis studies, we showed that selective NET and MAO inhibition increased DA levels in the PFC of wild‐type mice by two‐ to fourfold, an effect that was still doubled in COMT‐ko mice. Inhibition of DAT had no effect on DA levels in either genotype. Therefore, we conclude that in the mouse, PFC COMT contributes about one half of the total DA clearance.