The delayed rectifier Kv2.1 underlies the I_{k, slow} current in the rodent heart and is particularly interesting in that both its function and localization are regulated by many stimuli in neuronal systems. However, standard Immunolocalization approaches do not detect cardiac Kv2.1, therefore little is known regarding its localization in the heart. In the present study, we used recombinant adenovirus to determine the subcellular localization and lateral mobility of GFP-Kv2.1 and YFP-Kv1.4 in atrial and ventricular myocytes. In atrial myocytes, Kv2.1 formed large clusters on the cell surface similar to those observed in hippocampal neurons, whereas Kv1.4 was evenly distributed over both the peripheral sarcolemma and the transverse tubules. However, fluorescence recovery after photobleach (FRAP) experiments indicate that atrial Kv2.1 was immobile, while Kv1.4 was mobile ( = 252 ± 42 s). In ventricular myocytes, Kv2.1 did not form clusters and was localized primarily in the transverse-axial tubules and sarcolemma. In contrast, Kv1.4 was found only in transverse tubules and sarcolemma. FRAP studies revealed that Kv2.1 has a higher mobility in ventricular myocytes (= 479 ± 178 s), although its mobility is slower than Kv1.4 (1 = 18.9 ± 2.3 s; τ2 = 305 ± 55 s). We also observed the movement of small, intracellular transport vesicles containing GFP-Kv2.1 within ventricular myocytes. These data are the first evidence of Kv2.1 localization in living myocytes and indicate that Kv2.1 may have distinct physiological roles in atrial and ventricular myocytes.