The relative toxicity of numerous cardiotonic steroids (viz. ouabain, digitoxin, digoxin, convallatoxin, SC4453, bufalin, gitaloxin, digoxigenin, actodigin, oleandrin, digitoxigenin, gitoxin, strophanthidin, gitoxigenin, lanatosides A, B and C, alpha- and beta-acetyl digoxin, alpha- and beta-methyl digoxin) and related compounds towards a number of independent cell lines established from human, monkey, mouse, Syrian hamster, and Chinese hamster have been determined. All cardiac glycosides and their genins, as well as the cardiotonic alkaloid cassaine, exhibited greater than 100-fold higher toxicity towards cultured human and monkey cells in comparison to the cell lines of mouse, Syrian hamster, and Chinese hamster origins. These differences are species-related as all cell lines (both normal as well as transformed) from any one species, as well as cells from the closely related species (e.g., man and monkey or mouse, Chinese hamster, and Syrian hamster), showed similar sensitivity towards these drugs. The failure to see any significant differences in cellular toxicity for a larger number of other compounds which either bear limited structural resemblance to cardiac glycosides (viz. estradiol 17-beta-acetate, testosterone propionate, 21-acetoxy pregnenolone, beta-estradiol, digitonin, tigogenin, and tomatine) or interact with the Na+/K+ ATPase in a different manner (viz. veratridine, sanguinarine nitrate, penicillic acid, vanadium pentoxide, harmaline-HCI,5,5'-diphenyl hydantoin, quindonium bromide, and methyl quinolizinum bromide) provides strong evidence that the observed species-related differences are highly specific for cardiotonic steroids. Studies on the binding of [3H]ouabain show that, in comparison to human and monkey cell lines, no significant binding of the drug is observed in cells derived from the resistant species (i.e., mouse and Chinese hamster). The Na+/K+ ATPase from cells of the resistant species is inhibited at much higher concentrations of ouabain and digitoxin in comparison to the enzyme from human cells, and a good correlation is observed between these concentrations and those reported for inhibition of the enzyme from isolated heart muscles of the same species. These results provide strong evidence that the species-related differences in sensitivity to digitalis have a cellular basis and that the cultured cells from various mammalian species provide a useful model system for investigating the mechanism of action of cardiac glycosides.