14-3-3 proteins are dimeric switch proteins that modify the function of other proteins in a phosphorylation dependent manner. We have studied the mechanism by which 14-3-3 controls the trafficking of the dimeric K+ channels TASK-1 and TASK-3 using (i) voltage-clamp measurements after heterologous expression of channel mutants in Xenopus oocytes, (ii) imaging of GFP-tagged channels in COS-7 cells, and (iii) luminometric measurement of the surface expression of the reporter protein CD8 to which mutants of the TASK-1 C-terminus were attached. TASK-1 and -3 have a conserved "mode-3" 14-3-3 binding motif (RRxSx) at their C-termini, which is preceded by a tri-basic motif (in TASK-1, the motif is MKRRxSx). Impairment of 14-3-3 binding by removal of the last amino acid ([Delta]C1 mutant) caused retention of the channels in the endoplasmic reticulum. Removal of the tri-basic signal ([Delta]C5 mutant) partially rescued surface expression. Yeast-two-hybrid analysis showed that the [Delta]C1 mutant of the C-terminus of TASK-1 (but not the wild type C- terminus) interacted with components of the COPI coat. We conclude that binding of 14-3-3 enhances surface expression of TASK-1 and -3 by masking a tribasic retention signal. Since the binding of 14-3-3 is phosphorylation dependent, the surface expression of TASK-1 and -3 may be regulated post-translation- ally via second messengers by modulating the binding of 14-3-3.