Overexpression of p50/dynamitin, known to disrupt the dynein-dynactin complex responsible for carrying vesicle cargo, substantially increased outward K+ currents in HEK293 cells stably expressing Kv1.5 (0.57+0.07 nA/pF, n=12; to 1.18+0.2 nA/pF, n=12, p< 0.01), Kv4.2, and Kv2.1 channels, as did treatment of the cells with a dynamin inhibitory peptide, which blocks endocytosis. Nocodazole pretreatment, which depolymerizes the microtubule cytoskeleton along which dynein tracks, also doubled Kv1.5 currents in HEK cells and sustained K+ currents in isolated rat atrial myocytes. These increased currents were blocked by 1 mM 4-aminopyridine, and the specific Kv1.5 antagonist, DMM (100 nM). Confocal imaging of both HEK cells and myocytes, as well as experiments testing the sensitivity of the channel in living cells to external Proteinase K, showed that this increase in K+ current density was caused by a redistribution of channels toward the plasma membrane. Co-immunoprecipitation experiments demonstrated a direct interaction between Kv1.5 and the dynein motor complex in both heterologous cells and rat cardiac myocytes, supporting the role of this complex in Kv1.5 trafficking, which required an intact SH3-binding domain in the Kv1.5 N-terminus to occur. Throughout this post-internalization trafficking, the channels very probably reside in endosomes. As assayed by immunocytochemistry/confocal microscopy and by live-cell imaging, internalized Kv1.5 co-localized with both EEA1 and Rab5, markers of the early endosomal compartment, as well as with recycling endosome-specific Rab11. Furthermore, co-expression of Kv1.5 with a Rab5 dominant negative, which prevents early endosome maturation, increased Kv1.5 currents to an extent similar to that seen with p50 or the dynamin inhibitor. These experiments highlight a pathway for Kv1.5 internalization from the cell surface involving early endosomes, followed by later trafficking in endosomal vesicles by the dynein motor along microtubules. This work has significant implications for our understanding of the way Kv channel surface expression is regulated.