To identify proteins interacting with the C-terminal cytoplasmic tail of the dopamine D(3) receptor (D(3)R), we used the two-hybrid system to screen a rat brain cDNA library. We isolated three partial cDNAs encoding, respectively, for the MUPP1 multi-PDZ protein, for the N-terminal region of radixin, for GIPC and for a 160-amino acid open reading frame sharing high homology with the human CLIC6, also identified as parchorin in rabbit. In the two-hybrid system, CLIC6 was also able to interact with the D(2)R and D(4)R. The interaction between D(3)R and CLIC6 was confirmed by the use of a GST-D(3)R C-terminus fusion protein and COS cell extracts transiently expressing epitope-tagged CLIC6. In adult rat brain, CLIC6 mRNA expression was restricted to the choroid plexus, the striatal proliferative subventricular zone and the cerebellum where it is co-expressed with the D(3)R in the Purkinje cells of the lobules IX and X. CLIC6 mRNA was also detected in the pituitary in the posterior lobe and in cells co-expressing the D(2)R at the border between the intermediate and anterior lobes. In transfected HEK293 cells, D(3)R and CLIC6 co-localized at the plasma-membrane. No effect of CLIC6 transfection was observed on either intracellular chloride concentration or D(3)R/D(2)R-mediated response. In two-hybrid system, CLIC6 also interacted with MUPP1 and radixin but not GIPC, suggesting it could take part in a complex with D(2)-like receptors, not only by direct interaction with their C-termini, but also through interactions with scaffolding proteins.