The pharmacokinetics of the relatively polar and rapidly acting cardiac glycoside ouabain (G-strophanthin) were studied in dogs and human subjects by the use of a newly devised radioimmunoassay technic. This method had high specificity and a sensitivity of less than 0.1 ng of ouabain per ml of plasma or urine. After administration of a single intravenous dose, the plasma ouabain concentration fell rapidly in both dogs and humans. After 7 hours, a phase of exponential decline was reached which had a half-life of 18 hours in dogs and 21 hours in normal human subjects. Repeated intravenous administration of ouabain to human subjects for 9 consecutive days resulted in the establishment of a plateau of plasma concentration and urinary excretion after 4-5 days, confirming plasma and urinary half-lives in the 19-24-hour range. The mean ratio of renal clearance of ouabain to that of creatinine was 0.81 in the human subjects. The plasma half-life of ouabain determined in these studies is in good agreement with previous observations of the half-life of dissipation of positive inotropy and of slowing of ventricular response to atrial fibrillation after ouabain administration.