Little is known about the postinternalization trafficking of surface-expressed voltage-gated potassium channels. Here, for the first time, we investigate into which of four major trafficking pathways a voltage-gated potassium channel is targeted after internalization. In both a cardiac myoblast cell line and in HEK293 cells, channels were found to internalize and to recycle quickly. Upon internalization, Kv1.5 rapidly associated with Rab5-and Rab4-positive endosomes, suggesting that the channel is internalized via a Rab5-dependent pathway and rapidly targeted for recycling to the plasma membrane. Nevertheless, as indicated by colocalization with Rab7, a fraction of the channels are targeted for degradation. Recycling through perinuclear endosomes is limited; colocalization with Rab11 was evident only after 24 h postsurface labelling. Expression of dominant negative (DN) Rab constructs significantly increased Kv1.5 functional expression. In the myoblast line, Rab5DN increased Kv1.5 current densities to 1305 +/- 213 pA pF(-1) from control 675 +/- 81.6 pA pF(-1). Rab4DN similarly increased Kv1.5 currents to 1382 +/- 155 pA pF(-1) from the control 522 +/- 82.7 pA pF(-1) at +80 mV. Expression of the Rab7DN increased Kv1.5 currents 2.5-fold in HEK293 cells but had no significant effect in H9c2 myoblasts, and, unlike the other Rab GTPases tested, over-expression of wild-type Rab7 decreased Kv1.5 currents in the myoblast line. Densities fell to 573 +/- 96.3 pA pF(-1) from the control 869 +/- 135.5 pA pF(-1). The Rab11DN was slow to affect Kv1.5 currents but had comparable effects to other dominant negative constructs after 48 h. With the exception of Rab11DN and nocodazole, the effects of interference with microtubule-dependent trafficking by nocodazole or p50 overexpression were not additive with the Rab dominant negatives. The Rab GTPases thus constitute dynamic targets by which cells may modulate Kv1.5 functional expression.