A monoclonal antibody with a high affinity for digoxin (Ka = 1.14 X 10(9) M) was produced by somatic cell fusion. This antibody, designated 35/6.1, displayed little or no cross reactivity with other steroids. In the guinea-pig atria, 35/6.1 antagonized functionally the positive inotropic effect of digoxin but did not alter that of isoproterenol. Our results suggest that this monoclonal antibody may represent a useful probe for studying the physiological role of the putative endogenous digoxin-like factor in various pathophysiological conditions.