Adrenal medullary (AM) cells are exposed to high concentrations of cortical hormones, one of which is a ouabain-like substance. Thus, the effects of ouabain on catecholamine secretion and distribution of Na⁺+,K⁺-ATPase and subunits in rat and guinea-pig AM cells were examined using amperometry and immunological techniques. While exposure to 1 µM ouabain did not have a marked effect on resting secretion, it induced an increase in secretion due to mobilization of Ca²⁺ ions that were stored during a 4-min interval between muscarine applications. Immunocytochemistry revealed that Na⁺,K⁺-ATPase 1 subunit-like and 3 subunit-like immunoreactive (IR) materials were distributed ubiquitously at the cell periphery, whereas 2- and 2-like IR materials were present in restricted parts of the cell periphery. The 1 and 2 subunits were mainly immunoprecipitated from AM preparations by anti-3 and anti-2 antisera, respectively. Peripheral BODIPY-FL-InsP₃ binding sites were localized below membrane domains with 2- and 2-like IR materials. The results indicate that in AM cells, 13 isozymes of Na⁺,K⁺-ATPase were present ubiquitously in the plasma membrane, while 22 isozymes were in the membrane domain closely associated with peripheral Ca²⁺ store sites. This close association of the 22 isozyme with peripheral Ca²⁺ store sites may account for the facilitation of mobilization-dependent secretion in the presence of 1 µM ouabain.