Digoxin is the third most commonly prescribed drug in the United States and is routinely monitored in clinical chemistry laboratories. Polyclonal antisera used up until now in immunoassays for digoxin are not satisfactory and lead to poor precision and problems with standardization. Monoclonal antibodies would certainly be preferable because of high reproducibility and possibility of standardization which they ensure. Twenty-two anti-digoxin monoclonal antibodies (MoAbs) were selected by an ELISA absorption at the earliest stages of hybridoma growth. Dissociation constants of selected MoAbs were determined by ELISA. Detection limit in this system was also assessed. Specificity of twelve antibodies with sufficiently high-affinity constants to detect therapeutic and subtherapeutic levels of digoxin was determined by cross-reactivity experiments with 25 steroid compounds-cardiac glycosides, digoxin metabolites, steroid hormones, spironolactone. On the basis of these data, MoAbs with suitable binding parameters for immunodiagnostic purposes might be selected.