The extents of protein binding of 3H-digitoxin and 3H-digoxin in plasma of different species were determined in vitro by the equilibrium dialysis technique and liquid scintillation counting. Independence of drug concentration and the percentage of drug that was protein bound was observed for both digitoxin (0.01 to 1.00 μg/ml) and digoxin (0.0025 to 1.00 μg/ml). The extents of binding of 3H-digitoxin (50 ng/ml) and 3H-digoxin (10 ng/ml) were measured in goat, sheep, ox, horse, pony, swine, dog, cat, human, monkey, rat, rabbit and opossum plasmas. Digitoxin was more than 80% bound in all species except opossum (72%). There was a significant difference in extent of digitoxin binding among species (F-test, p