Well-controlled studies of the structural and functional interactions between cardiomyocytes and other cells are essential for understanding heart pathophysiology and for the further development of safe and efficient cell therapies. We established a novel in vitro assay comprised of a large number of individual micropatterned cell pairs with a reproducible shape, size, and region of cell-cell contact. This assay was applied to quantify and compare the frequency of expression and distribution of electrical (connexin-43) and mechanical (N-cadherin) coupling proteins in five thousand cell pairs made of cardiomyocytes (CMs), cardiac fibroblasts (CFs), skeletal myoblasts (SKMs), and mesenchymal stem cells (MSCs). We show that for all cell pair types, side-side contacts between two cells form 4.5-14.3 times more often than end-end contacts. Both connexin-43 and N-cadherin are expressed in all homotypic cardiomyocyte pairs, but in only 13.4%-91.6% pairs containing non-cardiomyocytes, where expression is either junctional (at the site of cell-cell contact) or diffuse (inside the cytoplasm). Cardiomyocyte expression is exclusively junctional in homotypic pairs but predominantly diffuse in heterotypic pairs. Non-cardiomyocyte homotypic pairs exhibit diffuse expression 1.7-8.7 times more often than junctional expression, which is increased 2.6-4.4 times in heterotypic pairs. Junctional connexin-43 and N-cadherin expression are respectively found in 38.6±7.3% and 39.6±6.2% of CM-MSC pairs, 21.9±5.0% and 13.6±1.9% of CM-SKM pairs, and in only 3.8-9.6% of CM-CF pairs. Measured frequencies of protein expression and distribution are stable for at least 4 days. Described studies in micropatterned cell pairs shed new light on cellular interactions relevant for cardiac function and cell therapies.