Calcium-sensitive potassium channels (KCa3.1) are expressed in virtually all migrating cells. Their activity is required for optimal cell migration so that their blockade leads to slowing down. KCa3.1 channels must be inserted into the plasma membrane in order to elicit their physiological function. However, the plasma membrane of migrating cells is subject to rapid recycling by means of endo- and exocytosis. Here, we focussed on the endocytic internalization and the intracellular transport of the human isoform hKCa3.1. A hKCa3.1 channel construct with an HA-tag in the extracellularly located S3-S4 linker was transfected into migrating transformed renal epithelial MDCK-F cells. Channel internalization was visualized and quantified with immunofluorescence and a cell-based ELISA. Movement of hKCa3.1 channel containing vesicles as well as migration of MDCK-F cells were monitored by means of time lapse video microscopy. hKCa3.1 channels are endocytosed during migration. Most of the hKCa3.1 channel containing vesicles are moving at a speed of up to 2 µm/sec in a microtubule-dependent manner towards the front of MDCK-F cells. Our experiments indicate that endocytosis of hKCa3.1 channels is clathrin-dependent since they colocalize with clathrin adaptor proteins and since it is impaired when a C-terminal dileucine motif is mutated. The C-terminal dileucine motif is also important for the subcellular localization of hKCa3.1 channels in migrating cells. Mutated channels are no longer concentrated at the leading edge. We therefore propose that recycling of hKCa3.1 channels contributes to their characteristic subcellular distribution in migrating cells. J. Cell. Physiol. 227: 686–696, 2012. © 2011 Wiley Periodicals, Inc.