Membrane-associated guanylate kinase proteins (MAGUKs) are important determinants of localization and organization of ion channels into specific plasma membrane domains. However, their exact role in channel function and cardiac excitability is not known. Here, we examined the effect of SAP97, a MAGUK abundantly expressed in the heart, on the function and localization of Kv1.5 subunits in cardiac myocytes. Recombinant SAP97 or Kv1.5 subunits tagged with GFP were overexpressed in rat neonatal cardiac myocytes and in CHO cells from adenoviral or plasmidic vectors. Immunocytochemistry, fluorescence recovery after photobleaching (FRAP) and patch-clamp techniques were used to study the effects of SAP97 on the localization, mobility and function of Kv1.5 subunits. Adenovirus-mediated SAP97 overexpression in cardiac myocytes resulted in the clustering of endogenous Kv1.5 subunits at myocyte-myocyte contacts and an increase in both the maintained component of the outward K⁺ current, I_Kur (5.64±0.57 pA/pF in SAP97 myocytes vs 3.23±0.43 pA/pF in controls) and the number of 4-AP-sensitive potassium channels in cell-attached membrane patches. In live myocytes, GFP-Kv1.5 subunits were mobile and organized in clusters at the basal plasma membrane whereas SAP97 overexpression reduced their mobility. In CHO cells, Kv1.5 channels were diffusely distributed throughout the cell body and freely mobile. When co-expressed with SAP97, Kv subunits were organized in plaque-like clusters and poorly mobile. In conclusion, SAP97 regulates the K⁺ current in cardiac myocytes by retaining and immobilizing Kv1.5 subunits in the plasma membrane. This new regulatory mechanism may contribute to the targeting of Kv channels in cardiac myocytes.