The Na+-K+-ATPase is specifically inhibited by cardiac glycosides, some of which may also function as endogenous mammalian hormones. Previous studies using Xenopus oocytes, yeast cells, or purified isoforms demonstrated that affinities of various cardiac glycosides for three isoforms of the Na+-K+-ATPase (α1-α3β1) may differ, a finding with potential clinical implication. The present study investigates isoform selectivity and effects of cardiac glycosides on cultured mammalian cells under more physiological conditions. H1299 cells (non-small cell lung carcinoma) were engineered to express only one α-isoform (α1, α2, or α3) by combining stable transfection of isoforms and silencing endogenous α1. Cardiac glycoside binding was measured by displacement of bound 3H-ouabain. The experiments confirm moderate α1/α3:α2 selectivity of ouabain, moderate α2:α1 selectivity of digoxin, and enhanced α2:α1 selectivity of synthetic derivatives (Katz A, Tal DM, Heller D, Haviv H, Rabah B, Barkana Y, Marcovich AL, Karlish SJD. J Biol Chem 289: 21153-21162, 2014). Relative α2:α1 selectivity of digoxin vs. ouabain was also manifested by enhanced internalization of α2 in response to digoxin. Cellular proliferation assays of H1299 cells confirmed the patterns of α2:α1 selectivity for ouabain, digoxin, and a synthetic derivative and reveal a crucial role of surface pump density on sensitivity to cardiac glycosides. Because cardiac glycosides are being considered as drugs for treatment of cancer, effects of ouabain on proliferation of 12 cancer and noncancer cell lines, with variable plasma membrane expression of α1, have been tested. These demonstrated that sensitivity to ouabain indeed depends linearly on the plasma membrane surface density of Na+-K+-ATPase irrespective of status, malignant or nonmalignant.