Voltage-gated sodium channel α and β subunits expressed in mammalian heart are differentially localized to t-tubules and intercalated disks. Sodium channel β subunits are multifunctional molecules that participate in channel modulation and cell adhesion. Reversible, receptor-mediated changes in β1 tyrosine phosphorylation modulate its ability to recruit and associate with ankyrin. The purpose of the present study was to test our hypothesis that tyrosine-phosphorylated β1 (pYβ1) and nonphosphorylated β1 subtmits may be differentially localized in heart and thus interact with different cytoskeletal and signaling proteins. We developed an antibody that specifically recognizes pYβ1 and investigated the differential subcellular localization of β1 and pYβ1 in mouse ventricular myocytes. We found that pYβ1 colocalized with connexin-43, N-cadherin, and Nav1.5 at intercalated disks but was not detected at the t-tubules. Anti-pYβ1 immunoprecipitates N-cadherin from heart membranes and from cells transfected with β1 and N-cadherin in the absence of other sodium channel subunits. pYβ1 does not associate with ankyrinB in heart membranes. N-cadherin and connexin-43 associate with Nav1.5 in heart membranes as assessed by co-immunoprecipitation assays. We propose that sodium channel complexes at intercalated disks of ventricular myocytes are composed of Nav1.5 and pYβ1 and that these complexes are in close association with both N-cadherin and connexin-43. β1 phosphorylation appears to regulate its localization to differential subcellular domains.