This is a contribution to the biological identification and estimation of the antidiuretic substance in human urine. The urine was concentrated and purified by column chromatography on Amberlite IRC-50 and by dialyses. It was possible to estimate the antidiuretic activity of this extract against arginine vasopressin by a fundamentally and statistically valid biological assay using water loaded rats anaesthetized with ethanol. Recovery experiments of vasopressin from urine at different steps of the purification were performed, and the final yield was estimated to be about 60 %. The amount of antidiuretic activity in the urine from four healthy subjects ranged between 11 mU and 24 mU/24 h with a mean of 16 mU/24 h.