Rats with hereditary hypothalamic diabetes insipidus, devoid of endogenous ADH, exhibited a prompt antidiuresis when injected subcutaneously or intraarterially with ovine prolactin. The antidiuresis was accompanied by a decrease in free water clearance and an increase in urine osmolality without a change in osmolal clearance or creatinine excretion. Measurement of PAH and insulin clearances indicated that prolactin had no effect on renal plasma flow or glomerular filtration rate. Prolactin injection caused a transient decrease in urinary sodium excretion, but proximal tubular sodium reabsorption, estimated by lissamine green transit time, was unaffected. The antidiuretic effect of prolactin could not be attributed to ADH contamination of the ovine prolactin preparation. Kidney cyclic AMP content was increased significantly 5 min after injection of prolactin. Thus, prolactin has an antidiuretic effect similar to that which occurs as a result of ADH action on the kidney and does not require either the release or the presence of ADH in order to cause the antidiuresis. Further, the impaired water excretion cannot be attributed to an increase in proximal tubular sodium reabsorption or to alteration of renal hemodynamics. It is suggested that prolactin has a direct ADH-like action on the kidney resulting in antidiuresis.