Although it is universally accepted that dopamine transporters (DATs) exist in monomers, dimers and tetramers (i.e. dimers of dimers), it is not known whether the oligomeric organization of DAT is a prerequisite for its ability to take up dopamine (DA), or whether each DAT protomer-the subunit of quaternary structure- functions independently in terms of DA translocation. In the present work copper phenanthroline (CuP) was used to selectively target surface DAT: increasing concentrations of CuP gradually cross-linked natural DAT dimers in LLC-PK1 cells stably expressing hDAT and thereby reduced DA uptake functionality until all surface DATs were inactivated. DATs that were not cross-linked by CuP showed normal DA uptake with DA Km at ~ 0.5 μM and DA efflux with basal and amphetamine-induced DA efflux as much as control values. The cocaine analog 2β-carbomethoxy-3β-[4-fluorophenyl]-tropane (CFT) was capable to bind to copper-cross-linked DATs, albeit with an affinity more than 5 fold decreased (Kd of CFT =109 nM after crosslinking vs 19 nM before). A kinetic analysis is offered describing the changing amounts of dimers and monomers with increasing [CuP], allowing the estimation of dimer functional activity compared with a DAT monomer. Consonant with previous conclusions for SERT and NET that only one protomer of an oligomer is active at the time, the present data indicated -a functional activity of the DAT dimer of 0.74 relative to a monomer. This article is protected by copyright. All rights reserved.